bst  (New England Biolabs)

Journal: BMC Genomics

Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

doi: 10.1186/1471-2164-7-312

Figure Lengend Snippet: Detection of Skp2 amplification in NSCLC samples following whole genome amplification by Bst DNA polymerase . The ratios of Skp2 to PIK3R1 gene were maintained in Bst amplified vs. non-amplified NSCLC samples. Error bars represent SD.

Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

Techniques: Amplification, Whole Genome Amplification

Journal: BMC Genomics

Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

doi: 10.1186/1471-2164-7-312

Figure Lengend Snippet: Gel electrophoresis of Bst DNA polymerase amplification products . From left to right: (1) Lambda DNA-Hind III digested ladder; FFPE samples: (2, 3) normal lung 3 4; (4, 5) neuroblastoma xenografts LAN-5 SK-N-BE (2) and (6, 7) NSCLC 3 4; (8) Commercial DNA; (9) Negative control. Samples were analyzed in 0.5% agarose gel, stained with SYBR-green II. 10% by volume of the amplification product was used for the gel electrophoresis.

Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

Techniques: Nucleic Acid Electrophoresis, Amplification, Lambda DNA Preparation, Formalin-fixed Paraffin-Embedded, Negative Control, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

Journal: BMC Genomics

Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

doi: 10.1186/1471-2164-7-312

Figure Lengend Snippet: N- myc gene content in Bst amplified vs. non-amplified neuroblastoma xenografts . For neuroblastoma xenografts, where N- myc gene is highly amplified, relative gene content in Bst amplified samples was comparable to the respective values in non-amplified samples and the representational distortion was negligible. Note: NBL-S is a neuroblastoma cell line that lacks N- myc amplification and appropriately the calculated copy numbers were 1.12 ± 0.03 for non-amplified DNA and 1.14 ± 0.35 for Bst amplified DNA. Error bars represent SD.

Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

Techniques: Amplification

Journal: BMC Genomics

Article Title: Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

doi: 10.1186/1471-2164-7-312

Figure Lengend Snippet: Mean amplification of DNA by Bst polymerase . All reactions started with 10 ng of target DNA. FFPE samples: Lung 1–5, neuroblastoma xenografts (LAN-5, NUB-7, SK-N-BE(2), NBL-S) and NSCLC 1–7. Intact DNA samples: FL (Frozen Lung) 1–4 and Positive C. (Control). Negative C. (Control) contained water in lieu of target DNA. For each sample the mean and SD of 2–6 independent experiments is shown.

Article Snippet: The reaction mixture was then brought up to 50 μL with 400 μM dNTPs in 1× ThermoPol buffer, 0.35 units/μL Bst DNA polymerase, large fragment (New England Biolabs, Ipswich, MA) and 4% final concentration of DMSO.

Techniques: Amplification, Formalin-fixed Paraffin-Embedded

Journal: Nucleic Acids Research

Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

doi: 10.1093/nar/gkp037

Figure Lengend Snippet: Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

Article Snippet: Fifteen microlitres of the adaptor ligated DNA were incubated for 30 min at 65°C in 1× ThermoPol Reaction Buffer (New England Biolabs), 8 µg Bovine Serum Albumin (BSA) (New England Biolabs), 20 nmol dNTPs and 16 U Bst DNA polymerase, Large Fragment (New England Biolabs) in a total volume of 20 µl.

Techniques: Isolation, In Vitro, Ligation, Generated, Magnetic Beads, Amplification, Nested PCR, Polymerase Chain Reaction, Software, Marker

Journal: Nucleic Acids Research

Article Title: Loop-mediated isothermal amplification of DNA

doi:

Figure Lengend Snippet: Detection of PSA mRNA by reverse transcription-coupled LAMP (RT-LAMP). Various numbers of LNCaP cells were mixed with 10 6 PSA-non-producing K562 cells and total RNA was extracted. RT-LAMP was carried out in the same reaction mixture as for M13mp18 DNA amplification except that 1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and 5 µg of extracted RNA were used. All the above components were mixed at once on ice and were incubated at 65°C for 45 min. The products were electrophoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-LAMP carried out in the presence and absence of Bst DNA polymerase or ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in lanes 6 and 7, respectively, but digested with Sau 3AI; lane M, 100 bp ladder (New England Biolabs).

Article Snippet: The mixture was heated at 95°C for 5 min, then chilled on ice, 8 U Bst DNA polymerase large fragment (New England Biolabs) were added, followed by incubation at 65°C for 1 h and heating at 80°C for 10 min to terminate the reaction.

Techniques: Amplification, Incubation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

Journal: Nucleic Acids Research

Article Title: Loop-mediated isothermal amplification of DNA

doi:

Figure Lengend Snippet: Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. ( A ) Electrophoretic analysis of the LAMP amplified M13mp18 product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2% agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with Pvu II; lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion with Bam HI, Pst I and Pvu II, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown in (A) was used for Southern blot hybridization with M13-281 DNA ( B ), M13-333 DNA ( C ) and M13BIP ( D ) as probes. ( E ) Alkaline agarose gel electrophoresis of the LAMP products. Lane m, λ DNA Hin dIII digests; lane 4, the same sample as in (A).

Article Snippet: The mixture was heated at 95°C for 5 min, then chilled on ice, 8 U Bst DNA polymerase large fragment (New England Biolabs) were added, followed by incubation at 65°C for 1 h and heating at 80°C for 10 min to terminate the reaction.

Techniques: Southern Blot, Hybridization, Amplification, Agarose Gel Electrophoresis, SYBR Green Assay, Staining, Marker

Journal: PLoS ONE

Article Title: Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli

doi: 10.1371/journal.pone.0230881

Figure Lengend Snippet: Amplification simulated diagram of the CPA and IMSA assays. A, Conditions and interval selection of assay optimization. B-C. Optimization of the incubation temperature, dNTPs concentration, Bst DNA polymerase concentration and incubation time for the CPA and IMSA assays, respectively. M: Standard DNA molecule marker; N: negative control. (The optimization items, corresponding conditions and units are marked in the figure, the best conditions highlighted with blue squares.).

Article Snippet: The CPA reaction was then performed at 63°C for 1 h and then terminated by incubation at 85°C for 3 min. For the IMSA assay, the reaction was also performed in a 30 μL mixture, which contained 1 μL (10 mM) outer primers DsF/DsR, 2.5 μL (10 mM) FIT/RIT and SteF/SteR, respectively, 3 μL 10 × Thermol-Pol reaction buffer, 2 μL (16 U) Bst DNA polymerase large fragment, 3 μL (1 mM) MgCl2 , 2 μL (0.8 M) betaine, 2.5 μL (1.0 mM) dNTPs and 2 μL of target DNA, the reaction system supplemented with sterilized deionized water.

Techniques: Amplification, Selection, Incubation, Concentration Assay, Marker, Negative Control