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  • 99
    Kapa Biosystems kapa taq dna polymerase
    Kapa Taq Dna Polymerase, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 99/100, based on 797 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs taq polymerase
    Amplification results of three target genes using <t>Taq</t> polymerase. (A) Attempted amplification of the three <t>DNA</t> targets ( mpb83 , Mb012, and LMHCC_RS00060 ) sequences by Taq in the presence of DMSO using 3St, 2St and TD protocols; mpb83 and LMHCC_RS00060 successfully showed amplicons using the three protocols. Mb012 failed to show an amplicon. (B) Attempts of amplification of the three DNA target sequences in the absence of DMSO using 3St, 2St and TD protocols showing no amplicon. mpb83 with added enhancer in 3St PCR is used as a positive control. Mb0129 with no enhancer in 3St PCR is used as a negative control.
    Taq Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8780 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taq dna polymerase
    Differential peak morphologies of androgen receptor alleles resulting from <t>DNA</t> dilution and reagent use. Lynx positive control DNA sample amplified with Invitrogen <t>Taq</t> DNA Polymerase and diluted to 1:10 ( a ), 1:20 ( b ), and 1:50 ( c ) ratios with deionized water. Lynx positive control DNA sample amplified with Invitrogen Platinum Taq DNA Polymerase (no dilution necessary) ( d )
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 44901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher platinum taq dna polymerase
    Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed <t>Taq</t> <t>DNA</t> polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.
    Platinum Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa taq dna polymerase
    (A) Genotype in each generation of mutant mice confirmed by PCR analysis on isolated genomic <t>DNA.</t> PCR was performed on the isolated genomic DNA using <t>taq</t> DNA polymerase and primer sets (#1 and #2 are for wild-type and GluR2 delta7 KI, #3 and #4 are for
    Taq Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 13199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega taq dna polymerase
    Superimposition of the thumb domains of <t>Taq</t> <t>DNA</t> polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
    Taq Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 19543 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa ex taq dna polymerase
    PCR analysis of the pgsA alleles of E. coli pgsA mutants. PCR products amplified with Ex <t>Taq</t> <t>DNA</t> polymerase were subjected to 1.2% agarose gel electrophoresis. Lanes 1, W3110; lanes 2, S301; lanes 3, MDL12; lanes 4 to 6, independent clones of the transductants (S330). Primer pairs FPPG5-ASFPPG1 (a) and FPPG5-ASFPPG3 (b) were used. The design and sequences of the primers are described in Materials and Methods. With the former primer pair, the wild-type pgsA allele and the pgsA :: kan allele gave products of 0.71 and 1.9 kbp, respectively. With the latter primer pair, the wild-type pgsA and the pgsA :: kan alleles gave products of 0.5 and 1.7 kbp, respectively. A DNA fragment of ca. 1.5 kbp which appeared in MDL12 (panel b, lane 3) may be the product of a false annealing of the antisense primer with a site in lacZ ′ fused to pgsA ). The molecular size markers included (two left lanes of each gel) were λ- Hin dIII digest (23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kbp) and λ- Eco T14 I digest (19.3, 7.7, 6.2, 4.3, 3.5, 2.7, 1.9, 1.5, 0.93, and 0.42 kbp).
    Ex Taq Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 9323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen taq dna polymerase
    Slowly migrating DNAs are converted into ocDNA by <t>Taq</t> (A) or T4 (B) <t>DNA</t> polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.
    Taq Dna Polymerase, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 9184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa la taq dna polymerase
    Optimization of the multiplex PCR components. (A) Annealing temperature gradients, M: DL2000 marker. Lanes 1–8: 64, 63, 62, 61, 60, 59, 58, and 57°C, respectively. (B) Dose of Anaplasma primers, lane M: DL2000 marker; Lanes 1–7: 0.08, 0.16, 0.24, 0.32, 0.4, 0.48, and 0.56 μM, respectively; Lane N: negative control. (C) La <t>Taq</t> <t>DNA</t> polymerase. M: DL2000 marker; Lanes 1–7: 0.75, 1.0, 1.25, 1.5, 1.75, 2, and 2.25 U, respectively; Lane N: negative control.
    La Taq Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 5238 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher platinum taq dna polymerase high fidelity
    RT-PCR analysis of the T HESCs cell line. (A) For the detection of the JAZF1/SUZ12 chimeric transcript, 1 μl cDNA was used as a template in PCR amplification with the primer combinations: human-JAZF1-284/Fusion-541R, Rhesus-JAZF1-284/Fusion-541R, human-JAZF1-286F/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R, the enzyme Platinum <t>Taq</t> <t>DNA</t> Polymerase High Fidelity and a touchdown PCR cycling protocol. Using the human-JAZF1-284/Fusion-541R and human-JAZF1-286F/Fusion-541R, JAZF1/SUZ12 chimeric transcripts were amplified in an endometrial stromal sarcoma carrying the t(7;17) chromosomal aberration [t(7;17)-ESS], whereas no JAZF1/SUZ12 chimeric transcripts were amplified in the T HESCs cell line. The Rhesus-JAZF1-284/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R did not generate any PCR products. (B) RT-PCR analysis showed that in the T HESCs cell line, both the normal JAZF1 and SUZ12 genes are expressed. The primer set for JAZF1 amplified a cDNA fragment that corresponds to the entire open reading of JAZF1 . The amplified cDNA fragment of SUZ12 contained part of exon 1, exons 2–6 and part of exon 7. M, 100-bp DNA ladder. Blank, no RNA in the cDNA synthesis.
    Platinum Taq Dna Polymerase High Fidelity, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore taq dna polymerase
    (A) The suggested effects of PtNPs on polymerase chain reaction (PCR) is based on binding of PtNPs to the <t>Taq</t> <t>DNA</t> polymerase, which leads to ceasing of PCR, (B) whereas CisPt primarily intercalates in DNA structure and stops PCR by this way. The gel electrophoregrams of PCR product mixture with particular concentration of (C) PtNPs (0.04–4 200 ng/mL of Pt) and (D) CisPt (0.04–42 000 ng/mL of Pt). (E) DNA denaturation temperature affected by the 0–200 μg/mL of Pt derivatives. Fluorescence of labelled nucleotides of DNA fragment after sequencing, which was influenced by (F) 0–20 μg/mL of PtNPs and (G) 0–0.33 μg/mL of CisPt. For all measurement n = 3.
    Taq Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3580 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare taq dna polymerase
    Incorporation enhancement attributable to terminator and polymerase variations. Incorporation is represented on a logarithmic scale relative to ddCTP incorporation by Vent <t>DNA</t> polymerase. Values for ThermoSequenase incorporation of ddCTP are inferred from Tabor and Richardson (20). <t>Taq</t> DNA polymerase failed to incorporate detectable amounts of acyCTP and thus is not represented. ROX analog incorporation was only examined for Vent and Vent A488L DNA polymerases.
    Taq Dna Polymerase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim taq dna polymerase
    (A) Agarose (2%) gel containing RT-PCR samples from heat-shocked and control cells. Lanes 1 and 2 contained control and heat-shocked RNA, respectively. Lanes 3 and 4 contained the RT products in lanes 1 and 2, respectively. Lane 5 contained the products resulting from PCR amplification of the material in lane 3. Lanes 6 and 7 contained parallel PCR mixtures from lane 4. Lanes c4 through c7 contained controls. Lane c4 contained the product resulting from PCR amplification of purified RNA (lanes 1 and 2). Lane c5 contained the product resulting from a complete RT-PCR performed with no initial template RNA. Lane c6 contained the RT-PCR product obtained without AMVRT, and lane c7 contained the PCR product resulting from RNA obtained without <t>DNA</t> <t>Taq</t> polymerase. (B) Agarose (2%) gel containing new RNA samples loaded in the same manner as those in panel A. However, the purified RNA samples were contaminated with DNA, as shown by the multiple bands in control lanes c4 and c6.
    Taq Dna Polymerase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 1740 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore jumpstart taq dna polymerase
    (A) Agarose (2%) gel containing RT-PCR samples from heat-shocked and control cells. Lanes 1 and 2 contained control and heat-shocked RNA, respectively. Lanes 3 and 4 contained the RT products in lanes 1 and 2, respectively. Lane 5 contained the products resulting from PCR amplification of the material in lane 3. Lanes 6 and 7 contained parallel PCR mixtures from lane 4. Lanes c4 through c7 contained controls. Lane c4 contained the product resulting from PCR amplification of purified RNA (lanes 1 and 2). Lane c5 contained the product resulting from a complete RT-PCR performed with no initial template RNA. Lane c6 contained the RT-PCR product obtained without AMVRT, and lane c7 contained the PCR product resulting from RNA obtained without <t>DNA</t> <t>Taq</t> polymerase. (B) Agarose (2%) gel containing new RNA samples loaded in the same manner as those in panel A. However, the purified RNA samples were contaminated with DNA, as shown by the multiple bands in control lanes c4 and c6.
    Jumpstart Taq Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Meridian Life Science taq dna polymerase
    (A) Agarose (2%) gel containing RT-PCR samples from heat-shocked and control cells. Lanes 1 and 2 contained control and heat-shocked RNA, respectively. Lanes 3 and 4 contained the RT products in lanes 1 and 2, respectively. Lane 5 contained the products resulting from PCR amplification of the material in lane 3. Lanes 6 and 7 contained parallel PCR mixtures from lane 4. Lanes c4 through c7 contained controls. Lane c4 contained the product resulting from PCR amplification of purified RNA (lanes 1 and 2). Lane c5 contained the product resulting from a complete RT-PCR performed with no initial template RNA. Lane c6 contained the RT-PCR product obtained without AMVRT, and lane c7 contained the PCR product resulting from RNA obtained without <t>DNA</t> <t>Taq</t> polymerase. (B) Agarose (2%) gel containing new RNA samples loaded in the same manner as those in panel A. However, the purified RNA samples were contaminated with DNA, as shown by the multiple bands in control lanes c4 and c6.
    Taq Dna Polymerase, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 93/100, based on 2073 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher accuprime taq dna polymerase
    (A) Agarose (2%) gel containing RT-PCR samples from heat-shocked and control cells. Lanes 1 and 2 contained control and heat-shocked RNA, respectively. Lanes 3 and 4 contained the RT products in lanes 1 and 2, respectively. Lane 5 contained the products resulting from PCR amplification of the material in lane 3. Lanes 6 and 7 contained parallel PCR mixtures from lane 4. Lanes c4 through c7 contained controls. Lane c4 contained the product resulting from PCR amplification of purified RNA (lanes 1 and 2). Lane c5 contained the product resulting from a complete RT-PCR performed with no initial template RNA. Lane c6 contained the RT-PCR product obtained without AMVRT, and lane c7 contained the PCR product resulting from RNA obtained without <t>DNA</t> <t>Taq</t> polymerase. (B) Agarose (2%) gel containing new RNA samples loaded in the same manner as those in panel A. However, the purified RNA samples were contaminated with DNA, as shown by the multiple bands in control lanes c4 and c6.
    Accuprime Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1644 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa titanium taq dna polymerase
    (A) Agarose (2%) gel containing RT-PCR samples from heat-shocked and control cells. Lanes 1 and 2 contained control and heat-shocked RNA, respectively. Lanes 3 and 4 contained the RT products in lanes 1 and 2, respectively. Lane 5 contained the products resulting from PCR amplification of the material in lane 3. Lanes 6 and 7 contained parallel PCR mixtures from lane 4. Lanes c4 through c7 contained controls. Lane c4 contained the product resulting from PCR amplification of purified RNA (lanes 1 and 2). Lane c5 contained the product resulting from a complete RT-PCR performed with no initial template RNA. Lane c6 contained the RT-PCR product obtained without AMVRT, and lane c7 contained the PCR product resulting from RNA obtained without <t>DNA</t> <t>Taq</t> polymerase. (B) Agarose (2%) gel containing new RNA samples loaded in the same manner as those in panel A. However, the purified RNA samples were contaminated with DNA, as shown by the multiple bands in control lanes c4 and c6.
    Titanium Taq Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1649 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega go taq dna polymerase
    H2A.B regulates the transcription of the imprinted Igf2r locus. ( A ) Schematic sketch of the Igf2r locus. ( B ) Retinoic acid (RA) induces the enrichment of H2A.B at the Igf2r locus. ChIP analysis on the Igf2r locus is performed in mouse ES cells with or without RA treatment. Data are presented as mean ± SEM ( n = 3). ( C , D ) H2A.B is deposited at the maternally methylated DMR of Igf2r following RA treatment. Allele-specific incorporation of H2A.B was determined by <t>DNA</t> sequencing ( C ) or PCR-SSCP ( D ) (see Methods). ( E ) Knockdown of H2A.B suppresses the incorporation of H2A.B at the DMR of Igf2r . ChIP analysis is performed in shRNA-treated ES cells with or without RA induction. Data are presented as mean ± SEM ( n = 3). ( F ) Knockdown of H2A.B suppresses the RA-induced transcription of Igf2r . The relative mRNA level of Igf2r is examined by qPCR. Data are presented as mean ± SEM ( n = 4). ( G ) Knockdown of H2A.B suppresses the transcription of Igf2r from the maternal allele following RA treatment. Left panel shows that maternal and paternal alleles are digested by <t>Taq</t> I into different patterns because of SNP. Right panel shows that the transcription of Igf2r is dependent on the maternally methylated allele following RA treatment, and that depletion of H2A.B suppresses the RA-induced transcription of Igf2r from the maternal allele. The reduction of the transcription of Igf2r from each allele following RA treatment is summarized in the histogram.
    Go Taq Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1472 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs longamp taq dna polymerase
    Thermal shock is crucial for successful PCR amplification from fungal spores. A. fumigatus spores at three different concentrations (8 × 10 8 /ml, 1 × 10 8 /ml, and 5 × 10 7 /ml) were prepared as described in Basic Protocol 1 . 3 µl of spore suspension were used as the <t>DNA</t> template for PCR. ( A ) PCR result from spore suspension subjected to thermal shock of 95°C for 15 min and −80°C for 10 min prior to PCR, as described in Basic Protocol 2 . No thermal shock was done for spore suspensions from Panels B and C . Subsequently, a PCR was run with an initial denaturation step of 95°C for 1 min (A), 5 min (B), or 15 min (C) with primers ITS1 and D2 (expected PCR product ∼1.2 kb) and <t>LongAmp</t> <t>Taq</t> DNA polymerase with PCR conditions described in Basic Protocol 2 . P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).
    Longamp Taq Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1023 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bangalore Genei taq dna polymerase
    Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start <t>Taq</t> <t>DNA</t> polymerase at same dilutions; lane M 100 bp marker
    Taq Dna Polymerase, supplied by Bangalore Genei, used in various techniques. Bioz Stars score: 94/100, based on 1139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CinnaGen Co taq dna polymerase
    Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start <t>Taq</t> <t>DNA</t> polymerase at same dilutions; lane M 100 bp marker
    Taq Dna Polymerase, supplied by CinnaGen Co, used in various techniques. Bioz Stars score: 92/100, based on 1418 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Amplification results of three target genes using Taq polymerase. (A) Attempted amplification of the three DNA targets ( mpb83 , Mb012, and LMHCC_RS00060 ) sequences by Taq in the presence of DMSO using 3St, 2St and TD protocols; mpb83 and LMHCC_RS00060 successfully showed amplicons using the three protocols. Mb012 failed to show an amplicon. (B) Attempts of amplification of the three DNA target sequences in the absence of DMSO using 3St, 2St and TD protocols showing no amplicon. mpb83 with added enhancer in 3St PCR is used as a positive control. Mb0129 with no enhancer in 3St PCR is used as a negative control.

    Journal: bioRxiv

    Article Title: PCR procedures to amplify GC-rich DNA sequences of Mycobacterium bovis

    doi: 10.1101/2020.02.18.953695

    Figure Lengend Snippet: Amplification results of three target genes using Taq polymerase. (A) Attempted amplification of the three DNA targets ( mpb83 , Mb012, and LMHCC_RS00060 ) sequences by Taq in the presence of DMSO using 3St, 2St and TD protocols; mpb83 and LMHCC_RS00060 successfully showed amplicons using the three protocols. Mb012 failed to show an amplicon. (B) Attempts of amplification of the three DNA target sequences in the absence of DMSO using 3St, 2St and TD protocols showing no amplicon. mpb83 with added enhancer in 3St PCR is used as a positive control. Mb0129 with no enhancer in 3St PCR is used as a negative control.

    Article Snippet: Unsuccessful attempts were faced with Taq polymerase, OneTaq DNA Polymerase (NEB, M0480S), Platinum™ Pfx DNA Polymerase (Invitrogen, 11708039), Expand Long Template PCR System (an enzyme mix that contains thermostable Taq DNA Polymerase and a thermostable DNA polymerase with proofreading activity, Roche, 11681834001).

    Techniques: Amplification, Polymerase Chain Reaction, Positive Control, Negative Control

    Differential peak morphologies of androgen receptor alleles resulting from DNA dilution and reagent use. Lynx positive control DNA sample amplified with Invitrogen Taq DNA Polymerase and diluted to 1:10 ( a ), 1:20 ( b ), and 1:50 ( c ) ratios with deionized water. Lynx positive control DNA sample amplified with Invitrogen Platinum Taq DNA Polymerase (no dilution necessary) ( d )

    Journal: BMC Genetics

    Article Title: A test of somatic mosaicism in the androgen receptor gene of Canada lynx (Lynx canadensis)

    doi: 10.1186/s12863-015-0284-y

    Figure Lengend Snippet: Differential peak morphologies of androgen receptor alleles resulting from DNA dilution and reagent use. Lynx positive control DNA sample amplified with Invitrogen Taq DNA Polymerase and diluted to 1:10 ( a ), 1:20 ( b ), and 1:50 ( c ) ratios with deionized water. Lynx positive control DNA sample amplified with Invitrogen Platinum Taq DNA Polymerase (no dilution necessary) ( d )

    Article Snippet: Amplification was conducted in a 10ul reaction containing deionized water (Invitrogen), 10X PCR Reaction Buffer (Invitrogen), 50 mM MgCl2 (Invitrogen), 100 mM dNTP solution (Invitrogen), 3 mg/mL BSA, 40uM forward and reverse primers (Integrated DNA Technologies) mentioned above (forward primers labeled with the fluorescent dye HEX), 0.0375U Invitrogen Taq DNA Polymerase, and 5 ng of DNA.

    Techniques: Positive Control, Amplification

    Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed Taq DNA polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Real-time one-step RT-PCR evaluation of cDNA priming strategies and the effect of unbalanced target concentrations . A) Real-time one-step RT-PCR evaluation of the effect of different RT primers on quantification of RNA expression in singleplex and in triplex. Reactions employed either an oligo(dT) 18 RT primer, a random decamer RT primer, or a combination of oligo(dT) 18 and random decamer RT primers for cDNA synthesis. In addition, each one-step RT-PCR protocol employed Taq DNA polymerase, M-MLV RT, 0.8 μg of human thymus total RNA, and CleanAmp™ Precision PCR primers. Reactions were performed in triplicate. B) Real-time one-step RT-PCR evaluation of triplex one-step RT-PCR amplifications using different custom prepared mixes containing three RNA standards in different ratios. The relative abundance for each mixture A through H is represented in the following format: (X:Y:Z), where the copies of the ABCA5 RNA standard is present at 10^X copies, the ABCA6 RNA standard is present at 10^Y copies, and the ABCA7 RNA standard is present at 10^Z copies. The observed copy number for each reaction, which was performed in triplicate, was obtained by extrapolation of the Cq to a standard curve for the ABCA5, ABCA6, and ABCA7 RNA standards. The resultant data for each RNA sample was normalized to ABCA7 and was plotted graphically.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Polymerase Chain Reaction

    One-step RT-PCR evaluation of unmodified and thermolabile CleanAmp™ Precision primers to amplify three different targets of ABCA transporters in singleplex, duplex, and triplex amplifications . For each gene of interest (ABCA5, ABCA6, and ABCA7), the PCR primers were unmodified or contained CleanAmp™ Precision modifications. Reverse transcription utilized an oligo(dT) 18 primer. Reactions contained Taq DNA polymerase, the appropriate reverse transcriptase, and 0.82 μg of human trachea total RNA. A) Reactions employed M-MLV reverse transcriptase and utilized an RT extension temperature of 42°C. B) Reactions employed SuperScript ® III reverse transcriptase (SSIII RT) and utilized an RT extension temperature of 55°C.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: One-step RT-PCR evaluation of unmodified and thermolabile CleanAmp™ Precision primers to amplify three different targets of ABCA transporters in singleplex, duplex, and triplex amplifications . For each gene of interest (ABCA5, ABCA6, and ABCA7), the PCR primers were unmodified or contained CleanAmp™ Precision modifications. Reverse transcription utilized an oligo(dT) 18 primer. Reactions contained Taq DNA polymerase, the appropriate reverse transcriptase, and 0.82 μg of human trachea total RNA. A) Reactions employed M-MLV reverse transcriptase and utilized an RT extension temperature of 42°C. B) Reactions employed SuperScript ® III reverse transcriptase (SSIII RT) and utilized an RT extension temperature of 55°C.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Incubation

    Singleplex and triplex real-time one-step RT-PCR detection of ABCA5, ABCA6, and ABCA7 in three different tissues . Reactions, which were performed in triplicate, contained M-MLV reverse transcriptase, an unmodified oligo(dT) 18 primer, Taq DNA polymerase and CleanAmp™ Precision PCR primers for the ABCA5, ABCA6 and ABCA7 genes. A standard curve for ABCA5, ABCA6, and ABCA7 was determined by employing ~10 1 to ~10 8 copies of the appropriate RNA standard. Each of the three human total RNA tissue samples (brain (0.78 μg), thymus (0.8 μg), and trachea (0.82 μg)) was amplified in singleplex and triplex format for detection of ABCA5, ABCA6, and ABCA7. The number of copies of each target in a given tissue was determined by extrapolating the resultant Cq values to the standard curve and normalizing the resultant values to the micrograms of input total RNA. A) The relative number of copies per microgram and standard deviation for each target in brain, thymus, and trachea total RNA is represented in a bar graph, which displays the results for singleplex and triplex amplifications. B) The corresponding agarose gel analysis of the three tissue samples amplified in singleplex and in triplex.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Singleplex and triplex real-time one-step RT-PCR detection of ABCA5, ABCA6, and ABCA7 in three different tissues . Reactions, which were performed in triplicate, contained M-MLV reverse transcriptase, an unmodified oligo(dT) 18 primer, Taq DNA polymerase and CleanAmp™ Precision PCR primers for the ABCA5, ABCA6 and ABCA7 genes. A standard curve for ABCA5, ABCA6, and ABCA7 was determined by employing ~10 1 to ~10 8 copies of the appropriate RNA standard. Each of the three human total RNA tissue samples (brain (0.78 μg), thymus (0.8 μg), and trachea (0.82 μg)) was amplified in singleplex and triplex format for detection of ABCA5, ABCA6, and ABCA7. The number of copies of each target in a given tissue was determined by extrapolating the resultant Cq values to the standard curve and normalizing the resultant values to the micrograms of input total RNA. A) The relative number of copies per microgram and standard deviation for each target in brain, thymus, and trachea total RNA is represented in a bar graph, which displays the results for singleplex and triplex amplifications. B) The corresponding agarose gel analysis of the three tissue samples amplified in singleplex and in triplex.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Standard Deviation, Agarose Gel Electrophoresis

    Evaluation of M-MLV and SSIII reverse transcriptases in multiplex one-step RT-PCR amplification of up to five targets . The amplification of increasing number of targets was evaluated by using either M-MLV RT (42°C) or SSIII RT (55°C). Reactions contained an oligo(dT) 18 primer, 0.82 μg of human trachea total RNA, CleanAmp™ Precision primers, and Taq DNA polymerase.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Evaluation of M-MLV and SSIII reverse transcriptases in multiplex one-step RT-PCR amplification of up to five targets . The amplification of increasing number of targets was evaluated by using either M-MLV RT (42°C) or SSIII RT (55°C). Reactions contained an oligo(dT) 18 primer, 0.82 μg of human trachea total RNA, CleanAmp™ Precision primers, and Taq DNA polymerase.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    (A) Genotype in each generation of mutant mice confirmed by PCR analysis on isolated genomic DNA. PCR was performed on the isolated genomic DNA using taq DNA polymerase and primer sets (#1 and #2 are for wild-type and GluR2 delta7 KI, #3 and #4 are for

    Journal: Neuroscience letters

    Article Title: Involvement of GluR2 and GluR3 subunit C-termini in the trigeminal spinal subnucleus caudalis and C1-C2 neurons in trigeminal neuropathic pain

    doi: 10.1016/j.neulet.2010.12.060

    Figure Lengend Snippet: (A) Genotype in each generation of mutant mice confirmed by PCR analysis on isolated genomic DNA. PCR was performed on the isolated genomic DNA using taq DNA polymerase and primer sets (#1 and #2 are for wild-type and GluR2 delta7 KI, #3 and #4 are for

    Article Snippet: PCR was performed on the isolated genomic DNA using taq DNA polymerase (TaKaRa Ex Taq™, Takara, Otsu, Japan) and primer sets (primer #21–23 for GluR2 delta7: #21, 5′-ACA GAG GAA GGT AGT GGA AGG GAG-3′; #22, 5′-CTT GGT TTG GTT GTT GGT CAT AGC-3′; #23, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′; primer #31–33 for GluR3 delta7: #31, 5′-CCA ATA CTC CAC AGG GGC AAT TTA TC-3′; #32, 5′-CCG TTG ACT GTT TTG AAT CTC ACA CC-3′; #33, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′).

    Techniques: Mutagenesis, Mouse Assay, Polymerase Chain Reaction, Isolation

    Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

    Article Snippet: Thioredoxin stimulates the activity of Taq DNA pol/TBD by ∼4-fold and Taq DNA pol/TBD(exo–) enzymatic activities by > 30-fold but had no effect on Taq DNA polymerase.

    Techniques: Sequencing

    Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

    Article Snippet: Thioredoxin stimulates the activity of Taq DNA pol/TBD by ∼4-fold and Taq DNA pol/TBD(exo–) enzymatic activities by > 30-fold but had no effect on Taq DNA polymerase.

    Techniques: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

    Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

    Article Snippet: Thioredoxin stimulates the activity of Taq DNA pol/TBD by ∼4-fold and Taq DNA pol/TBD(exo–) enzymatic activities by > 30-fold but had no effect on Taq DNA polymerase.

    Techniques: Incubation

    The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

    Article Snippet: Thioredoxin stimulates the activity of Taq DNA pol/TBD by ∼4-fold and Taq DNA pol/TBD(exo–) enzymatic activities by > 30-fold but had no effect on Taq DNA polymerase.

    Techniques: Activity Assay, Labeling, Concentration Assay

    PCR analysis of the pgsA alleles of E. coli pgsA mutants. PCR products amplified with Ex Taq DNA polymerase were subjected to 1.2% agarose gel electrophoresis. Lanes 1, W3110; lanes 2, S301; lanes 3, MDL12; lanes 4 to 6, independent clones of the transductants (S330). Primer pairs FPPG5-ASFPPG1 (a) and FPPG5-ASFPPG3 (b) were used. The design and sequences of the primers are described in Materials and Methods. With the former primer pair, the wild-type pgsA allele and the pgsA :: kan allele gave products of 0.71 and 1.9 kbp, respectively. With the latter primer pair, the wild-type pgsA and the pgsA :: kan alleles gave products of 0.5 and 1.7 kbp, respectively. A DNA fragment of ca. 1.5 kbp which appeared in MDL12 (panel b, lane 3) may be the product of a false annealing of the antisense primer with a site in lacZ ′ fused to pgsA ). The molecular size markers included (two left lanes of each gel) were λ- Hin dIII digest (23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kbp) and λ- Eco T14 I digest (19.3, 7.7, 6.2, 4.3, 3.5, 2.7, 1.9, 1.5, 0.93, and 0.42 kbp).

    Journal: Journal of Bacteriology

    Article Title: Viability of an Escherichia coli pgsA Null Mutant Lacking Detectable Phosphatidylglycerol and Cardiolipin

    doi:

    Figure Lengend Snippet: PCR analysis of the pgsA alleles of E. coli pgsA mutants. PCR products amplified with Ex Taq DNA polymerase were subjected to 1.2% agarose gel electrophoresis. Lanes 1, W3110; lanes 2, S301; lanes 3, MDL12; lanes 4 to 6, independent clones of the transductants (S330). Primer pairs FPPG5-ASFPPG1 (a) and FPPG5-ASFPPG3 (b) were used. The design and sequences of the primers are described in Materials and Methods. With the former primer pair, the wild-type pgsA allele and the pgsA :: kan allele gave products of 0.71 and 1.9 kbp, respectively. With the latter primer pair, the wild-type pgsA and the pgsA :: kan alleles gave products of 0.5 and 1.7 kbp, respectively. A DNA fragment of ca. 1.5 kbp which appeared in MDL12 (panel b, lane 3) may be the product of a false annealing of the antisense primer with a site in lacZ ′ fused to pgsA ). The molecular size markers included (two left lanes of each gel) were λ- Hin dIII digest (23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kbp) and λ- Eco T14 I digest (19.3, 7.7, 6.2, 4.3, 3.5, 2.7, 1.9, 1.5, 0.93, and 0.42 kbp).

    Article Snippet: DNA from fresh colonies of the strains to be examined was used as a template for PCR amplification with Ex Taq DNA polymerase (Takara, Tokyo, Japan).

    Techniques: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Clone Assay

    Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.

    Journal: Journal of Virology

    Article Title: Transgenically Expressed T-Rep of Tomato Yellow Leaf Curl Sardinia Virus Acts as a trans-Dominant-Negative Mutant, Inhibiting Viral Transcription and Replication

    doi: 10.1128/JVI.75.22.10573-10581.2001

    Figure Lengend Snippet: Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.

    Article Snippet: To verify the nature of these slowly migrating molecules, TNAs extracted from the samples at 24 and 72 h were incubated with Taq DNA polymerase and analyzed by Southern blot (Fig. B).

    Techniques: Incubation, Transgenic Assay, Infection, Migration

    Slowly migrating DNAs also accumulate at early stages after transfection of wt protoplasts with TYLCSV. TNAs were extracted from protoplasts transfected with pTOM6. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slow-migrating viral DNAs are indicated with an asterisk (∗). HPT, hours posttransfection. (A) Time course analysis of viral DNA forms. Sample at 72 h was diluted 20-fold to give signals of satisfactory intensity on the autoradiographic film. (B) Taq DNA polymerase treatment of TNAs at 24 and 72 h after transfection. Samples were incubated for 10 min at 37°C with (+) or without (−) the polymerase. Lane C, sample at 72 h digested with Bgl II to show the linear DNA.

    Journal: Journal of Virology

    Article Title: Transgenically Expressed T-Rep of Tomato Yellow Leaf Curl Sardinia Virus Acts as a trans-Dominant-Negative Mutant, Inhibiting Viral Transcription and Replication

    doi: 10.1128/JVI.75.22.10573-10581.2001

    Figure Lengend Snippet: Slowly migrating DNAs also accumulate at early stages after transfection of wt protoplasts with TYLCSV. TNAs were extracted from protoplasts transfected with pTOM6. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slow-migrating viral DNAs are indicated with an asterisk (∗). HPT, hours posttransfection. (A) Time course analysis of viral DNA forms. Sample at 72 h was diluted 20-fold to give signals of satisfactory intensity on the autoradiographic film. (B) Taq DNA polymerase treatment of TNAs at 24 and 72 h after transfection. Samples were incubated for 10 min at 37°C with (+) or without (−) the polymerase. Lane C, sample at 72 h digested with Bgl II to show the linear DNA.

    Article Snippet: To verify the nature of these slowly migrating molecules, TNAs extracted from the samples at 24 and 72 h were incubated with Taq DNA polymerase and analyzed by Southern blot (Fig. B).

    Techniques: Transfection, Incubation

    Optimisation of the PPLMD method . The y-axis represents the average background-subtracted pixel density (0–64,000). A. Introduction of SpikeTarget DNA reduced background in negative control (purple vs. yellow). Blue: genomic DNA (containing RRS and MON810) + SpikeTarget, purple: MQ, yellow: MQ with SpikeTarget. All 4 padlock probes were present in the mix. HotStar Taq DNA polymerase was used to amplify in 40 LATE cycles. B. 80 LATE cycles increased signal compared to 40 cycles. Purple: 40 LATE cycles, blue: 80 LATE cycles. All 4 padlock probes were present in the mix (containing RRS and MON810). Vent ® exo - DNA polymerase was used.

    Journal: BMC Genomics

    Article Title: Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction

    doi: 10.1186/1471-2164-9-584

    Figure Lengend Snippet: Optimisation of the PPLMD method . The y-axis represents the average background-subtracted pixel density (0–64,000). A. Introduction of SpikeTarget DNA reduced background in negative control (purple vs. yellow). Blue: genomic DNA (containing RRS and MON810) + SpikeTarget, purple: MQ, yellow: MQ with SpikeTarget. All 4 padlock probes were present in the mix. HotStar Taq DNA polymerase was used to amplify in 40 LATE cycles. B. 80 LATE cycles increased signal compared to 40 cycles. Purple: 40 LATE cycles, blue: 80 LATE cycles. All 4 padlock probes were present in the mix (containing RRS and MON810). Vent ® exo - DNA polymerase was used.

    Article Snippet: The results showed a tendency that positive signals were improved and background signals were decreased compared to the use of HotStar Taq DNA polymerase, although statistical evidence is lacking (results not shown).

    Techniques: Negative Control

    Effect of individual constituents of the PCR on background fluorescence in the presence or absence of CABS. Each column represents the result of three PCRs performed: 1, in the absence of Taq Gold polymerase, but in the presence of oligonucleotides and template; 2, in the absence of Taq Gold polymerase and template, but in the presence of oligonucleotides; 3, in the absence of Taq Gold polymerase, template, and one oligonucleotide but in the presence of one oligonucleotide; 4, in the absence of primers and template, but in the presence of Taq Gold polymerase; 5, in the absence of template, but in the presence of both primers and polymerase; 6, as in case 3, except in the presence of the alternative primer of the primer combination; 7, in which specific amplicon is produced; and 8, in which no amplicon is expected. Conditions 1, 2, and 6 show the effect of CABS in reducing the background fluorescence caused by the single-strand oligonucleotides. The reaction with CABS in conditions 5 and 8 shows the residual effect caused by primer/dimers, in the case of 8 with additional template DNA.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Alkaline-mediated differential interaction (AMDI): A simple automatable single-nucleotide polymorphism assay

    doi: 10.1073/pnas.041619998

    Figure Lengend Snippet: Effect of individual constituents of the PCR on background fluorescence in the presence or absence of CABS. Each column represents the result of three PCRs performed: 1, in the absence of Taq Gold polymerase, but in the presence of oligonucleotides and template; 2, in the absence of Taq Gold polymerase and template, but in the presence of oligonucleotides; 3, in the absence of Taq Gold polymerase, template, and one oligonucleotide but in the presence of one oligonucleotide; 4, in the absence of primers and template, but in the presence of Taq Gold polymerase; 5, in the absence of template, but in the presence of both primers and polymerase; 6, as in case 3, except in the presence of the alternative primer of the primer combination; 7, in which specific amplicon is produced; and 8, in which no amplicon is expected. Conditions 1, 2, and 6 show the effect of CABS in reducing the background fluorescence caused by the single-strand oligonucleotides. The reaction with CABS in conditions 5 and 8 shows the residual effect caused by primer/dimers, in the case of 8 with additional template DNA.

    Article Snippet: The reaction mixture consisted of 67 mM Tris⋅HCl (pH 8.4) at 25°C, 16.6 mM ammonium sulfate, 2 mM magnesium chloride, 0.01% (vol/vol) Tween 20, 200 μM of each dNTP, 1 μM of each primer, and 0.5 units Hotstart Taq DNA polymerase (Qiagen) or AmpliTaq Gold (Perkin–Elmer).

    Techniques: Polymerase Chain Reaction, Fluorescence, Amplification, Produced

    Optimization of the multiplex PCR components. (A) Annealing temperature gradients, M: DL2000 marker. Lanes 1–8: 64, 63, 62, 61, 60, 59, 58, and 57°C, respectively. (B) Dose of Anaplasma primers, lane M: DL2000 marker; Lanes 1–7: 0.08, 0.16, 0.24, 0.32, 0.4, 0.48, and 0.56 μM, respectively; Lane N: negative control. (C) La Taq DNA polymerase. M: DL2000 marker; Lanes 1–7: 0.75, 1.0, 1.25, 1.5, 1.75, 2, and 2.25 U, respectively; Lane N: negative control.

    Journal: Frontiers in Microbiology

    Article Title: A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood Samples

    doi: 10.3389/fmicb.2020.00606

    Figure Lengend Snippet: Optimization of the multiplex PCR components. (A) Annealing temperature gradients, M: DL2000 marker. Lanes 1–8: 64, 63, 62, 61, 60, 59, 58, and 57°C, respectively. (B) Dose of Anaplasma primers, lane M: DL2000 marker; Lanes 1–7: 0.08, 0.16, 0.24, 0.32, 0.4, 0.48, and 0.56 μM, respectively; Lane N: negative control. (C) La Taq DNA polymerase. M: DL2000 marker; Lanes 1–7: 0.75, 1.0, 1.25, 1.5, 1.75, 2, and 2.25 U, respectively; Lane N: negative control.

    Article Snippet: Optimization of the Multiplex PCR After optimization, the optimum multiplex PCR assay was performed in a final volume of 25 μL, containing 2.5 μL of 10× PCR La buffer, 4 μL of dNTPs at 2.5 mM, 1.25 U of La Taq DNA polymerase, 0.32 μM of each primer, and 2 μL of the DNA template.

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Marker, Negative Control

    RT-PCR analysis of the T HESCs cell line. (A) For the detection of the JAZF1/SUZ12 chimeric transcript, 1 μl cDNA was used as a template in PCR amplification with the primer combinations: human-JAZF1-284/Fusion-541R, Rhesus-JAZF1-284/Fusion-541R, human-JAZF1-286F/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R, the enzyme Platinum Taq DNA Polymerase High Fidelity and a touchdown PCR cycling protocol. Using the human-JAZF1-284/Fusion-541R and human-JAZF1-286F/Fusion-541R, JAZF1/SUZ12 chimeric transcripts were amplified in an endometrial stromal sarcoma carrying the t(7;17) chromosomal aberration [t(7;17)-ESS], whereas no JAZF1/SUZ12 chimeric transcripts were amplified in the T HESCs cell line. The Rhesus-JAZF1-284/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R did not generate any PCR products. (B) RT-PCR analysis showed that in the T HESCs cell line, both the normal JAZF1 and SUZ12 genes are expressed. The primer set for JAZF1 amplified a cDNA fragment that corresponds to the entire open reading of JAZF1 . The amplified cDNA fragment of SUZ12 contained part of exon 1, exons 2–6 and part of exon 7. M, 100-bp DNA ladder. Blank, no RNA in the cDNA synthesis.

    Journal: Oncology Letters

    Article Title: Absence of the JAZF1/SUZ12 chimeric transcript in the immortalized non-neoplastic endometrial stromal cell line T HESCs

    doi: 10.3892/ol.2010.185

    Figure Lengend Snippet: RT-PCR analysis of the T HESCs cell line. (A) For the detection of the JAZF1/SUZ12 chimeric transcript, 1 μl cDNA was used as a template in PCR amplification with the primer combinations: human-JAZF1-284/Fusion-541R, Rhesus-JAZF1-284/Fusion-541R, human-JAZF1-286F/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R, the enzyme Platinum Taq DNA Polymerase High Fidelity and a touchdown PCR cycling protocol. Using the human-JAZF1-284/Fusion-541R and human-JAZF1-286F/Fusion-541R, JAZF1/SUZ12 chimeric transcripts were amplified in an endometrial stromal sarcoma carrying the t(7;17) chromosomal aberration [t(7;17)-ESS], whereas no JAZF1/SUZ12 chimeric transcripts were amplified in the T HESCs cell line. The Rhesus-JAZF1-284/Fusion-541R and Rhesus-JAZF1-286F/Fusion-541R did not generate any PCR products. (B) RT-PCR analysis showed that in the T HESCs cell line, both the normal JAZF1 and SUZ12 genes are expressed. The primer set for JAZF1 amplified a cDNA fragment that corresponds to the entire open reading of JAZF1 . The amplified cDNA fragment of SUZ12 contained part of exon 1, exons 2–6 and part of exon 7. M, 100-bp DNA ladder. Blank, no RNA in the cDNA synthesis.

    Article Snippet: For the detection of the JAZF1/SUZ12 fusion transcript, PCR was performed with Platinum Taq DNA Polymerase High Fidelity (Invitrogen).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Touchdown PCR

    Analysis of the recombinant ANGPTL7/CDT6 plasmid (pNC1) in primary human trabecular meshwork cells. Two primary HTM cell lines (HTM-55 and HTM-69) were nucleofector-transfected with either ANGPTL7/CDT6 plasmid DNA or mock-transfected, and assayed at 72-h post-transfection. (A) Normalized ANGPTL7/CDT6 cDNA in the treated cells versus the mock-transfected cells. Top panel : representative C T logarithmic curve of the hybridizations of ANGPTL7/CDT6 and endogenous 18S cDNAs from treated and mock-transfected cells with their corresponding Taq Man probes. Bottom panel : fold change of ANGPTL7/CDT6 cDNA in treated versus mock-transfected, normalized to 18S and expressed as fold change mean ± range ( n = 3 * P

    Journal: Genes to cells : devoted to molecular & cellular mechanisms

    Article Title: Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma

    doi: 10.1111/j.1365-2443.2010.01483.x

    Figure Lengend Snippet: Analysis of the recombinant ANGPTL7/CDT6 plasmid (pNC1) in primary human trabecular meshwork cells. Two primary HTM cell lines (HTM-55 and HTM-69) were nucleofector-transfected with either ANGPTL7/CDT6 plasmid DNA or mock-transfected, and assayed at 72-h post-transfection. (A) Normalized ANGPTL7/CDT6 cDNA in the treated cells versus the mock-transfected cells. Top panel : representative C T logarithmic curve of the hybridizations of ANGPTL7/CDT6 and endogenous 18S cDNAs from treated and mock-transfected cells with their corresponding Taq Man probes. Bottom panel : fold change of ANGPTL7/CDT6 cDNA in treated versus mock-transfected, normalized to 18S and expressed as fold change mean ± range ( n = 3 * P

    Article Snippet: PCR was carried out in a 50-μL reaction mixture containing 5 μL 10× high-fidelity PCR buffer, 1 μL dNTP (10 μM each), 2 μL MgSO4 (50 μM), 4 μL primers (5 μM each), 1 μL template cDNA and 1 μL Platinum® Taq High Fidelity DNA Polymerase (5 U) (Invitrogen).

    Techniques: Recombinant, Plasmid Preparation, Transfection

    (A) The suggested effects of PtNPs on polymerase chain reaction (PCR) is based on binding of PtNPs to the Taq DNA polymerase, which leads to ceasing of PCR, (B) whereas CisPt primarily intercalates in DNA structure and stops PCR by this way. The gel electrophoregrams of PCR product mixture with particular concentration of (C) PtNPs (0.04–4 200 ng/mL of Pt) and (D) CisPt (0.04–42 000 ng/mL of Pt). (E) DNA denaturation temperature affected by the 0–200 μg/mL of Pt derivatives. Fluorescence of labelled nucleotides of DNA fragment after sequencing, which was influenced by (F) 0–20 μg/mL of PtNPs and (G) 0–0.33 μg/mL of CisPt. For all measurement n = 3.

    Journal: PLoS ONE

    Article Title: Platinum nanoparticles induce damage to DNA and inhibit DNA replication

    doi: 10.1371/journal.pone.0180798

    Figure Lengend Snippet: (A) The suggested effects of PtNPs on polymerase chain reaction (PCR) is based on binding of PtNPs to the Taq DNA polymerase, which leads to ceasing of PCR, (B) whereas CisPt primarily intercalates in DNA structure and stops PCR by this way. The gel electrophoregrams of PCR product mixture with particular concentration of (C) PtNPs (0.04–4 200 ng/mL of Pt) and (D) CisPt (0.04–42 000 ng/mL of Pt). (E) DNA denaturation temperature affected by the 0–200 μg/mL of Pt derivatives. Fluorescence of labelled nucleotides of DNA fragment after sequencing, which was influenced by (F) 0–20 μg/mL of PtNPs and (G) 0–0.33 μg/mL of CisPt. For all measurement n = 3.

    Article Snippet: The volume of the reaction mixture was 25 μL, which was composed of 2.5 μL of 10× standard Taq reaction buffer, 0.5 μL of 1 mM deoxynucleotide solution, 0.5 μL of each of the primers (10 μM), 0.125 μL of Taq DNA polymerase; selected volume of water or drugs diluted with water (sterile, ACS purity, Sigma-Aldrich) and 0.5 μL of bacteriophage λ DNA.

    Techniques: Polymerase Chain Reaction, Binding Assay, Concentration Assay, Fluorescence, Sequencing

    Incorporation enhancement attributable to terminator and polymerase variations. Incorporation is represented on a logarithmic scale relative to ddCTP incorporation by Vent DNA polymerase. Values for ThermoSequenase incorporation of ddCTP are inferred from Tabor and Richardson (20). Taq DNA polymerase failed to incorporate detectable amounts of acyCTP and thus is not represented. ROX analog incorporation was only examined for Vent and Vent A488L DNA polymerases.

    Journal: Nucleic Acids Research

    Article Title: Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases

    doi:

    Figure Lengend Snippet: Incorporation enhancement attributable to terminator and polymerase variations. Incorporation is represented on a logarithmic scale relative to ddCTP incorporation by Vent DNA polymerase. Values for ThermoSequenase incorporation of ddCTP are inferred from Tabor and Richardson (20). Taq DNA polymerase failed to incorporate detectable amounts of acyCTP and thus is not represented. ROX analog incorporation was only examined for Vent and Vent A488L DNA polymerases.

    Article Snippet: Taq DNA polymerase (Amersham Pharmacia Biotech, Piscataway, NJ) and its F667Y derivative Thermo Sequenase (Amersham Pharmacia Biotech) naturally lack this activity, while the archaeon DNA polymerases have been genetically modified within the conserved exonuclease motif DIE to AIA to eliminate this activity ( – ).

    Techniques:

    Incorporation of ddCTP and acyCTP. Titration assay for the indicated terminators using ( A ) Taq ( B ) Vent, ( C ) ThermoSequenase or ( D ) Vent A488L DNA polymerase. Extension of a 32 P-labeled primer on a M13mp18 single-stranded substrate was examined in the presence of the indicated ratios of terminator to dNTP. The lane marked dNTP is a control reaction performed in the absence of terminators.

    Journal: Nucleic Acids Research

    Article Title: Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases

    doi:

    Figure Lengend Snippet: Incorporation of ddCTP and acyCTP. Titration assay for the indicated terminators using ( A ) Taq ( B ) Vent, ( C ) ThermoSequenase or ( D ) Vent A488L DNA polymerase. Extension of a 32 P-labeled primer on a M13mp18 single-stranded substrate was examined in the presence of the indicated ratios of terminator to dNTP. The lane marked dNTP is a control reaction performed in the absence of terminators.

    Article Snippet: Taq DNA polymerase (Amersham Pharmacia Biotech, Piscataway, NJ) and its F667Y derivative Thermo Sequenase (Amersham Pharmacia Biotech) naturally lack this activity, while the archaeon DNA polymerases have been genetically modified within the conserved exonuclease motif DIE to AIA to eliminate this activity ( – ).

    Techniques: Titration, Labeling

    (A) Agarose (2%) gel containing RT-PCR samples from heat-shocked and control cells. Lanes 1 and 2 contained control and heat-shocked RNA, respectively. Lanes 3 and 4 contained the RT products in lanes 1 and 2, respectively. Lane 5 contained the products resulting from PCR amplification of the material in lane 3. Lanes 6 and 7 contained parallel PCR mixtures from lane 4. Lanes c4 through c7 contained controls. Lane c4 contained the product resulting from PCR amplification of purified RNA (lanes 1 and 2). Lane c5 contained the product resulting from a complete RT-PCR performed with no initial template RNA. Lane c6 contained the RT-PCR product obtained without AMVRT, and lane c7 contained the PCR product resulting from RNA obtained without DNA Taq polymerase. (B) Agarose (2%) gel containing new RNA samples loaded in the same manner as those in panel A. However, the purified RNA samples were contaminated with DNA, as shown by the multiple bands in control lanes c4 and c6.

    Journal: Applied and Environmental Microbiology

    Article Title: Reverse Transcription-PCR Differential Display Analysis of Escherichia coli Global Gene Regulation in Response to Heat Shock

    doi:

    Figure Lengend Snippet: (A) Agarose (2%) gel containing RT-PCR samples from heat-shocked and control cells. Lanes 1 and 2 contained control and heat-shocked RNA, respectively. Lanes 3 and 4 contained the RT products in lanes 1 and 2, respectively. Lane 5 contained the products resulting from PCR amplification of the material in lane 3. Lanes 6 and 7 contained parallel PCR mixtures from lane 4. Lanes c4 through c7 contained controls. Lane c4 contained the product resulting from PCR amplification of purified RNA (lanes 1 and 2). Lane c5 contained the product resulting from a complete RT-PCR performed with no initial template RNA. Lane c6 contained the RT-PCR product obtained without AMVRT, and lane c7 contained the PCR product resulting from RNA obtained without DNA Taq polymerase. (B) Agarose (2%) gel containing new RNA samples loaded in the same manner as those in panel A. However, the purified RNA samples were contaminated with DNA, as shown by the multiple bands in control lanes c4 and c6.

    Article Snippet: Each reaction mixture contained Taq DNA polymerase reaction buffer (10 mM Tris HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2 )(Boehringer Mannheim), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dTTP, 0.5 mM dGTP (all deoxynucleoside triphosphates were obtained from Boehringer Mannheim), each RT primer (Life Technologies) at a concentration of 0.5 μM, each PCR primer (Life Technologies) at a concentration of 0.5 μM, 0.05 U of Taq DNA polymerase (Boehringer Mannheim) per μl, 5 μl of cDNA, and enough autoclaved deionized water to bring the total volume to 60 μl.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Purification

    H2A.B regulates the transcription of the imprinted Igf2r locus. ( A ) Schematic sketch of the Igf2r locus. ( B ) Retinoic acid (RA) induces the enrichment of H2A.B at the Igf2r locus. ChIP analysis on the Igf2r locus is performed in mouse ES cells with or without RA treatment. Data are presented as mean ± SEM ( n = 3). ( C , D ) H2A.B is deposited at the maternally methylated DMR of Igf2r following RA treatment. Allele-specific incorporation of H2A.B was determined by DNA sequencing ( C ) or PCR-SSCP ( D ) (see Methods). ( E ) Knockdown of H2A.B suppresses the incorporation of H2A.B at the DMR of Igf2r . ChIP analysis is performed in shRNA-treated ES cells with or without RA induction. Data are presented as mean ± SEM ( n = 3). ( F ) Knockdown of H2A.B suppresses the RA-induced transcription of Igf2r . The relative mRNA level of Igf2r is examined by qPCR. Data are presented as mean ± SEM ( n = 4). ( G ) Knockdown of H2A.B suppresses the transcription of Igf2r from the maternal allele following RA treatment. Left panel shows that maternal and paternal alleles are digested by Taq I into different patterns because of SNP. Right panel shows that the transcription of Igf2r is dependent on the maternally methylated allele following RA treatment, and that depletion of H2A.B suppresses the RA-induced transcription of Igf2r from the maternal allele. The reduction of the transcription of Igf2r from each allele following RA treatment is summarized in the histogram.

    Journal: Genome Research

    Article Title: H2A.B facilitates transcription elongation at methylated CpG loci

    doi: 10.1101/gr.156877.113

    Figure Lengend Snippet: H2A.B regulates the transcription of the imprinted Igf2r locus. ( A ) Schematic sketch of the Igf2r locus. ( B ) Retinoic acid (RA) induces the enrichment of H2A.B at the Igf2r locus. ChIP analysis on the Igf2r locus is performed in mouse ES cells with or without RA treatment. Data are presented as mean ± SEM ( n = 3). ( C , D ) H2A.B is deposited at the maternally methylated DMR of Igf2r following RA treatment. Allele-specific incorporation of H2A.B was determined by DNA sequencing ( C ) or PCR-SSCP ( D ) (see Methods). ( E ) Knockdown of H2A.B suppresses the incorporation of H2A.B at the DMR of Igf2r . ChIP analysis is performed in shRNA-treated ES cells with or without RA induction. Data are presented as mean ± SEM ( n = 3). ( F ) Knockdown of H2A.B suppresses the RA-induced transcription of Igf2r . The relative mRNA level of Igf2r is examined by qPCR. Data are presented as mean ± SEM ( n = 4). ( G ) Knockdown of H2A.B suppresses the transcription of Igf2r from the maternal allele following RA treatment. Left panel shows that maternal and paternal alleles are digested by Taq I into different patterns because of SNP. Right panel shows that the transcription of Igf2r is dependent on the maternally methylated allele following RA treatment, and that depletion of H2A.B suppresses the RA-induced transcription of Igf2r from the maternal allele. The reduction of the transcription of Igf2r from each allele following RA treatment is summarized in the histogram.

    Article Snippet: All PCRs except those for region C of Kcnq1 were carried out with Go-Taq DNA polymerase (Promega) using 0.3 μM of each primer (Supplemental Table S2) and 0.1 μCi of [32 P]dCTP.

    Techniques: Chromatin Immunoprecipitation, Methylation, DNA Sequencing, Polymerase Chain Reaction, shRNA, Real-time Polymerase Chain Reaction

    Thermal shock is crucial for successful PCR amplification from fungal spores. A. fumigatus spores at three different concentrations (8 × 10 8 /ml, 1 × 10 8 /ml, and 5 × 10 7 /ml) were prepared as described in Basic Protocol 1 . 3 µl of spore suspension were used as the DNA template for PCR. ( A ) PCR result from spore suspension subjected to thermal shock of 95°C for 15 min and −80°C for 10 min prior to PCR, as described in Basic Protocol 2 . No thermal shock was done for spore suspensions from Panels B and C . Subsequently, a PCR was run with an initial denaturation step of 95°C for 1 min (A), 5 min (B), or 15 min (C) with primers ITS1 and D2 (expected PCR product ∼1.2 kb) and LongAmp Taq DNA polymerase with PCR conditions described in Basic Protocol 2 . P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

    Journal: Current Protocols in Microbiology

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction

    doi: 10.1002/cpmc.89

    Figure Lengend Snippet: Thermal shock is crucial for successful PCR amplification from fungal spores. A. fumigatus spores at three different concentrations (8 × 10 8 /ml, 1 × 10 8 /ml, and 5 × 10 7 /ml) were prepared as described in Basic Protocol 1 . 3 µl of spore suspension were used as the DNA template for PCR. ( A ) PCR result from spore suspension subjected to thermal shock of 95°C for 15 min and −80°C for 10 min prior to PCR, as described in Basic Protocol 2 . No thermal shock was done for spore suspensions from Panels B and C . Subsequently, a PCR was run with an initial denaturation step of 95°C for 1 min (A), 5 min (B), or 15 min (C) with primers ITS1 and D2 (expected PCR product ∼1.2 kb) and LongAmp Taq DNA polymerase with PCR conditions described in Basic Protocol 2 . P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).

    Article Snippet: Materials Spore suspension (Basic Protocol ) Primers (5 µM each, forward and reverse; experiment specific) LongAmp Taq DNA polymerase with 5× reaction buffer (New England Biolabs; M0323) dNTP mix (5 mM of each dNTP) Sterile, molecular‐grade water For agarose gel electrophoresis (also see Current Protocols article: Voytas, ): 6× DNA loading dye 1× TAE buffer (see recipe) Ethidium bromide or other DNA gel stain 0.2‐ml PCR tubes, 0.2‐ml/well 96‐well PCR plates, or 150 µl/well 384 well PCR plates with tight (preferably aluminum) seals Thermal cycler Centrifuge UV transilluminator Additional reagents and equipment for agarose gel electrophoresis (see Current Protocols article: Voytas, )

    Techniques: Polymerase Chain Reaction, Amplification, Negative Control

    Spore PCR using the supernatant from spore suspensions of different filamentous fungi with primers ITS1/D2 (expected PCR band size is ∼1.2 kb) and ITS1/ITS4 (expected PCR band size ∼600 bp). Two different spore concentrations were tested (i.e., 5 × 10 7 /ml and 1 × 10 7 /ml). 1 µl of the supernatant was used in the PCR reaction with the LongAmp Taq DNA polymerase. Positive PCR controls were amplified from DNA (50 ng) of the A. fumigatus wild‐type strain with primers ITS1/D2 (P1) and ITS1/ITS4 (P2). N: negative control (no DNA).

    Journal: Current Protocols in Microbiology

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction

    doi: 10.1002/cpmc.89

    Figure Lengend Snippet: Spore PCR using the supernatant from spore suspensions of different filamentous fungi with primers ITS1/D2 (expected PCR band size is ∼1.2 kb) and ITS1/ITS4 (expected PCR band size ∼600 bp). Two different spore concentrations were tested (i.e., 5 × 10 7 /ml and 1 × 10 7 /ml). 1 µl of the supernatant was used in the PCR reaction with the LongAmp Taq DNA polymerase. Positive PCR controls were amplified from DNA (50 ng) of the A. fumigatus wild‐type strain with primers ITS1/D2 (P1) and ITS1/ITS4 (P2). N: negative control (no DNA).

    Article Snippet: Materials Spore suspension (Basic Protocol ) Primers (5 µM each, forward and reverse; experiment specific) LongAmp Taq DNA polymerase with 5× reaction buffer (New England Biolabs; M0323) dNTP mix (5 mM of each dNTP) Sterile, molecular‐grade water For agarose gel electrophoresis (also see Current Protocols article: Voytas, ): 6× DNA loading dye 1× TAE buffer (see recipe) Ethidium bromide or other DNA gel stain 0.2‐ml PCR tubes, 0.2‐ml/well 96‐well PCR plates, or 150 µl/well 384 well PCR plates with tight (preferably aluminum) seals Thermal cycler Centrifuge UV transilluminator Additional reagents and equipment for agarose gel electrophoresis (see Current Protocols article: Voytas, )

    Techniques: Polymerase Chain Reaction, Amplification, Negative Control

    Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start Taq DNA polymerase at same dilutions; lane M 100 bp marker

    Journal: Indian Journal of Microbiology

    Article Title: Simultaneous Detection of Major Pome Fruit Viruses and a Viroid

    doi: 10.1007/s12088-013-0431-y

    Figure Lengend Snippet: Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start Taq DNA polymerase at same dilutions; lane M 100 bp marker

    Article Snippet: For RT, all conditions were the same except random hexamer primers 100 ng were used for cDNA synthesis and for multiplex PCR reaction 5 μl cDNA, 0.16 pmol of each primers pair, 1.2× Taq buffer, 0.3 mM dNTPs, 1.5 units of Taq DNA polymerase (Bangalore Genei) and 1.5 mM MgCl2 (additional) in final concentration was used. cDNA was amplified for 35 cycles (40 s at 94 °C, 75 s at 58 °C, and 90 s at 72 °C).

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Multiplexing, Marker