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PrimerDesign Inc complimentary dna cdna
Complimentary Dna Cdna, supplied by PrimerDesign Inc, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 87 stars, based on 2 article reviews
Price from $9.99 to $1999.99
complimentary dna cdna - by Bioz Stars, 2020-01
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Article Snippet: One microgram total RNA was converted to complementary DNA (cDNA) using the prescription NanoScript reverse transcription kit (RT – NanoScript, Primer Design), according to the manufacturer's instruction. cDNA was stored at −20 °C until further use.

Article Title:
Article Snippet: RNA isolated from T. b. brucei B48 strains and Leishmania species was quantified using a NanoDrop device; 2 μg of RNA was diluted in RNase-free water to a total volume of 25 μl 200 ng of RNA from each generated and control cell line, were used for the production of complementary DNA (cDNA) using a Reverse-Transcriptase (RT) kit (Primerdesign, UK).

Article Title:
Article Snippet: Complementary DNA (cDNA) was then synthesized using the Precision nanoScript 2 Reverse Transcription Kit (PrimerDesign, catalogue number RT‐nanoscript2) with a 1 : 1 mixture of random nonamer and Oligo‐dT primers, according to the manufacturer's instructions.

Article Title:
Article Snippet: The enzyme was deactivated by heating at 55 °C for 5 min. mRNA was reverse-transcribed (1 μg per reaction) into complementary DNA (cDNA) using a Precision nanoScript cDNA Reverse Transcription kit (Primer Design).

Article Title:
Article Snippet: Complimentary DNA (cDNA) was synthesised according to kit manufacturer protocols (PrimerDesign Ltd, UK).

Article Title:
Article Snippet: For all samples, total RNA was extracted and directly reverse-transcribed to complementary DNA (cDNA) using Precision qScript RT Kit (Primer Design).

Article Title:
Article Snippet: Complementary DNA (cDNA) was produced using a ReverseTranscriptase (RT) kit (Primerdesign, UK). cDNA for each sample was diluted 1∶10 and then used for Real Time-PCR.

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    PrimerDesign Inc cdna
    Characterisation of VEGF isoform expression in mouse and human <t>cDNA</t> extracts using VEGFxxxb isoform-specific RT-PCR. A , RT-PCR using an exon 4 forward primer (DB exon4) and an exon8 b/7 isoform-specific reverse primer (DB165b/189b-1) failed to detect any PCR products in commercial human tissue <t>cDNAs</t> (AMS Biotechnology Ltd.). B C , RT-PCR using an exon 3 forward primer and a reverse primer designed to amplify VEGF164/188 (164b/188b-1) detected a single PCR product in mouse fibrosarcoma cell lines, as well as a number of PCR products in solid fibrosarcomas and normal CBA and SCID mouse tissues. Product a represents a truncated VEGF species containing exons 3, 4 8b. Product b represents a product identical in sequence to VEGF164b. Product c represents a product exhibiting 100% identity to murine acetyl co-A acetyl transferase. D , No products corresponding to VEGF120b were detected in mouse cDNAs when RT-PCR was performed using the same exon 3 forward primer but a reverse primer designed to amplify VEGF120b (120b-1).
    Cdna, supplied by PrimerDesign Inc, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdna/product/PrimerDesign Inc
    Average 95 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    cdna - by Bioz Stars, 2020-01
    95/100 stars
      Buy from Supplier

    95
    PrimerDesign Inc cdna synthesis
    Generation of recq2 null mutants in bloodstream form T. brucei . ( A ) PCR was used to amplify a 5’ and a 3’ region (blue arrows) of the RECQ2 ORF (orange arrow). These PCR products were the ‘targeting regions’ cloned into the constructs shown below,which weremade to generate heterozygous (+/-) and knockout (-/-) mutants by replacing the ORF with resistance cassettes. Generalised representation of the linearised blasticidin and neomycin (G418) resistance constructs relative to the RECQ2 ORF (orange) and the targeting flanking regions (UTR; grey), which allow HR-mediated exchange (crosses). β/α tub, β/ α tubulin intergenic region; actin IR, actin intergenic region; BSD , blasticidin resistance; NEO , neomycin (G418) resistance. Not to scale. ( B ) The upper diagram shows diagnostic PCRs to confirm replacement of RECQ2 with the knockout constructs (positions of primers are indicted by arrows): a region of the ORF was amplified (“ORF PCR”) and, in addition, a region was amplified using a forward primer lying upstream of the 5’ UTR region in the knockout construct and a reverse primer specific to the BSD or NEO genes (“BSD PCR” and “NEO PCR”, respectively). The lower diagram shows agarose gels of PCR products generated from genomic DNA from wild type (WT) cells and heterozygote (+/-) and knockout (-/-) clones using primers described above. Distilled water was used as a negative control (-). Broken lines indicate different images aligned in this figure; size markers are shown (bp). ( C ). RT-PCR confirmation of recq2 mutants. A region of the PIF6 ORF and a region of the RECQ2 ORF was PCR-amplified using <t>cDNA</t> (+RT) synthesised from <t>RNA</t> extracted from wild type (wt) cells and from recq2 +/- and recq2 -/- mutants; control samples in which no reverse transcriptase had been added (-RT) are shown, as are controls using distilled water rather than substrate (-); size markers on the agarose gels are shown (bp). DOI: http://dx.doi.org/10.7554/eLife.12765.004
    Cdna Synthesis, supplied by PrimerDesign Inc, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdna synthesis/product/PrimerDesign Inc
    Average 95 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    cdna synthesis - by Bioz Stars, 2020-01
    95/100 stars
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    Characterisation of VEGF isoform expression in mouse and human cDNA extracts using VEGFxxxb isoform-specific RT-PCR. A , RT-PCR using an exon 4 forward primer (DB exon4) and an exon8 b/7 isoform-specific reverse primer (DB165b/189b-1) failed to detect any PCR products in commercial human tissue cDNAs (AMS Biotechnology Ltd.). B C , RT-PCR using an exon 3 forward primer and a reverse primer designed to amplify VEGF164/188 (164b/188b-1) detected a single PCR product in mouse fibrosarcoma cell lines, as well as a number of PCR products in solid fibrosarcomas and normal CBA and SCID mouse tissues. Product a represents a truncated VEGF species containing exons 3, 4 8b. Product b represents a product identical in sequence to VEGF164b. Product c represents a product exhibiting 100% identity to murine acetyl co-A acetyl transferase. D , No products corresponding to VEGF120b were detected in mouse cDNAs when RT-PCR was performed using the same exon 3 forward primer but a reverse primer designed to amplify VEGF120b (120b-1).

    Journal: PLoS ONE

    Article Title: Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

    doi: 10.1371/journal.pone.0035231

    Figure Lengend Snippet: Characterisation of VEGF isoform expression in mouse and human cDNA extracts using VEGFxxxb isoform-specific RT-PCR. A , RT-PCR using an exon 4 forward primer (DB exon4) and an exon8 b/7 isoform-specific reverse primer (DB165b/189b-1) failed to detect any PCR products in commercial human tissue cDNAs (AMS Biotechnology Ltd.). B C , RT-PCR using an exon 3 forward primer and a reverse primer designed to amplify VEGF164/188 (164b/188b-1) detected a single PCR product in mouse fibrosarcoma cell lines, as well as a number of PCR products in solid fibrosarcomas and normal CBA and SCID mouse tissues. Product a represents a truncated VEGF species containing exons 3, 4 8b. Product b represents a product identical in sequence to VEGF164b. Product c represents a product exhibiting 100% identity to murine acetyl co-A acetyl transferase. D , No products corresponding to VEGF120b were detected in mouse cDNAs when RT-PCR was performed using the same exon 3 forward primer but a reverse primer designed to amplify VEGF120b (120b-1).

    Article Snippet: PCR reactions were routinely performed in 25 µl using 1 µl of cDNA (AMS cDNAs) and 20 µl using 5 µl of cDNA (Primer Design cDNAs).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Affinity Magnetic Separation, Crocin Bleaching Assay, Sequencing

    A general RT-PCR approach using primers to exon 3 or 4 and 3'UTR failed to detect VEGFxxxb isoforms in mouse and human cDNA extracts respectively. A , PCR products corresponding to VEGF188 (474 bp), VEGF164 (402 bp), VEGF164/120 heteroduplex (arrowed) and VEGF120 (270 bp) are evident in the panel of mouse cDNAs amplified using the 3′UTR reverse primer and a forward primer to exon 3. B C , PCR products corresponding to VEGF189 (371 bp), VEGF165 (299 bp) and VEGF121 (167 bp) are evident in the commercial human tissue cDNAs amplified using the 3′UTR reverse primer (DB 3'UTR) and a forward primer to exon 4 (DB exon4). D , Amplification of the heteroduplex species (arrowed) a lso occurred when VEGF164 and VEGF120 tumour cDNAs were pooled and analysed by RT-PCR using exon3/3'UTR primers (lanes labelled 120+164). E , The same heteroduplex species was generated when cDNAs from VEGF164 and VEGF188 expressing fibrosarcoma cells were pooled and amplified as above (lanes labelled 120+164). M corresponds to a 100 bp ladder, whilst -tem represents a control PCR reaction in which water was used instead of cDNA template. The Figure contains data we published previously [25] .

    Journal: PLoS ONE

    Article Title: Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

    doi: 10.1371/journal.pone.0035231

    Figure Lengend Snippet: A general RT-PCR approach using primers to exon 3 or 4 and 3'UTR failed to detect VEGFxxxb isoforms in mouse and human cDNA extracts respectively. A , PCR products corresponding to VEGF188 (474 bp), VEGF164 (402 bp), VEGF164/120 heteroduplex (arrowed) and VEGF120 (270 bp) are evident in the panel of mouse cDNAs amplified using the 3′UTR reverse primer and a forward primer to exon 3. B C , PCR products corresponding to VEGF189 (371 bp), VEGF165 (299 bp) and VEGF121 (167 bp) are evident in the commercial human tissue cDNAs amplified using the 3′UTR reverse primer (DB 3'UTR) and a forward primer to exon 4 (DB exon4). D , Amplification of the heteroduplex species (arrowed) a lso occurred when VEGF164 and VEGF120 tumour cDNAs were pooled and analysed by RT-PCR using exon3/3'UTR primers (lanes labelled 120+164). E , The same heteroduplex species was generated when cDNAs from VEGF164 and VEGF188 expressing fibrosarcoma cells were pooled and amplified as above (lanes labelled 120+164). M corresponds to a 100 bp ladder, whilst -tem represents a control PCR reaction in which water was used instead of cDNA template. The Figure contains data we published previously [25] .

    Article Snippet: PCR reactions were routinely performed in 25 µl using 1 µl of cDNA (AMS cDNAs) and 20 µl using 5 µl of cDNA (Primer Design cDNAs).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Generated, Expressing, Transmission Electron Microscopy

    Discriminating VEGF isoform-specific RT-PCR using human cDNA extracts further highlights the importance of primer design. A , Human cDNAs from colon, prostate and kidney (Primer Design Ltd) were amplified in RT-PCR reactions using an exon 4 forward primer together with either DB165b/189b-1 (lane 1), h165b/189b-2 (lane 2), h165b/189b-3 (lane 3), h165b/189b-4 (lane 4) or h165b/189b-5 (lane 5). Primer sequences are highl ighted in Table 1 Fig 2 . A putative VEGF165b (∼212 bp) product is evident in lanes 2 3, but absent in lane 1. All three reverse primers spanned 5 bases across the 8b/7 junction, however differed in their melting temperatures (see main text), with h165b/189b-2 3 exhibiting more compatible Tms with the forward primer. A similar 165b product was more abundantly amplified in lanes 4 5 using reverse primers spanning more bases (13 or 14 respectively) across the 8b/7 splice junction. In addition, these latter reverse primers also amplified a product corresponding to 188b (∼283 bp). However DNA sequencing revealed that the product amplified in lanes 2 3 was not VEGF but LIM domain only, protein isoform 7, confirming that detection of apparent VEGFxxxb species was only evident using 8b/7 reverse primers with increasing complementary sequence to exon7. B , Similar results were obtained in RT-PCR reactions using cDNA extracted from a normal kidney patient biopsy and amplified with the exon 4 forward primer and the DB165b/189b-1 (lane1), h165b/189b-2 (lane 2) and h165b/189b-4 (lane 4) reverse primers. C , RT-PCR using the DBexon7/DB3'UTR forward/reverse primers capable of simultaneously amplifying VEGFxxx VEGFxxxb, confirmed only VEGFxxx (194 bp) amplification in the human samples tested.

    Journal: PLoS ONE

    Article Title: Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

    doi: 10.1371/journal.pone.0035231

    Figure Lengend Snippet: Discriminating VEGF isoform-specific RT-PCR using human cDNA extracts further highlights the importance of primer design. A , Human cDNAs from colon, prostate and kidney (Primer Design Ltd) were amplified in RT-PCR reactions using an exon 4 forward primer together with either DB165b/189b-1 (lane 1), h165b/189b-2 (lane 2), h165b/189b-3 (lane 3), h165b/189b-4 (lane 4) or h165b/189b-5 (lane 5). Primer sequences are highl ighted in Table 1 Fig 2 . A putative VEGF165b (∼212 bp) product is evident in lanes 2 3, but absent in lane 1. All three reverse primers spanned 5 bases across the 8b/7 junction, however differed in their melting temperatures (see main text), with h165b/189b-2 3 exhibiting more compatible Tms with the forward primer. A similar 165b product was more abundantly amplified in lanes 4 5 using reverse primers spanning more bases (13 or 14 respectively) across the 8b/7 splice junction. In addition, these latter reverse primers also amplified a product corresponding to 188b (∼283 bp). However DNA sequencing revealed that the product amplified in lanes 2 3 was not VEGF but LIM domain only, protein isoform 7, confirming that detection of apparent VEGFxxxb species was only evident using 8b/7 reverse primers with increasing complementary sequence to exon7. B , Similar results were obtained in RT-PCR reactions using cDNA extracted from a normal kidney patient biopsy and amplified with the exon 4 forward primer and the DB165b/189b-1 (lane1), h165b/189b-2 (lane 2) and h165b/189b-4 (lane 4) reverse primers. C , RT-PCR using the DBexon7/DB3'UTR forward/reverse primers capable of simultaneously amplifying VEGFxxx VEGFxxxb, confirmed only VEGFxxx (194 bp) amplification in the human samples tested.

    Article Snippet: PCR reactions were routinely performed in 25 µl using 1 µl of cDNA (AMS cDNAs) and 20 µl using 5 µl of cDNA (Primer Design cDNAs).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, DNA Sequencing, Sequencing

    A general RT-PCR approach with primers to exon 7a and 3'UTR failed to detect VEGFxxxb isoforms in human and mouse cDNA extracts. RT-PCR reactions were performed using forward/reverse primers designed to amplify both VEGFxxx and VEGFxxxb isoforms from cDNAs extracted from mouse fibrosarcoma cell lines tumours and normal mouse tissues (heart, lung, liver and kidney) as well as commercially sourced human tissue cDNAs. A , A single product corresponding to VEGF164/188 (194 bp) is evident in the panel of mouse cDNAs using forward and reverse primers designed to exon 7 and the 3'UTR (exon7/3′UTR) with no evidence of VEGF164b/188b (128 bp) products. B C , A single product corresponding to VEGF165/189 (194 bp) is evident in human brain, bladder and kidney (AMS Biotechnology Ltd), and prostate and kidney (Primer Design Ltd) using forward and reverse primers designed to exon 7 (DB exon7a) and the 3'UTR (DB 3'UTR) respectively. No products corresponding to VEGF165b/189b (128 bp) are evident in any of these samples.

    Journal: PLoS ONE

    Article Title: Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

    doi: 10.1371/journal.pone.0035231

    Figure Lengend Snippet: A general RT-PCR approach with primers to exon 7a and 3'UTR failed to detect VEGFxxxb isoforms in human and mouse cDNA extracts. RT-PCR reactions were performed using forward/reverse primers designed to amplify both VEGFxxx and VEGFxxxb isoforms from cDNAs extracted from mouse fibrosarcoma cell lines tumours and normal mouse tissues (heart, lung, liver and kidney) as well as commercially sourced human tissue cDNAs. A , A single product corresponding to VEGF164/188 (194 bp) is evident in the panel of mouse cDNAs using forward and reverse primers designed to exon 7 and the 3'UTR (exon7/3′UTR) with no evidence of VEGF164b/188b (128 bp) products. B C , A single product corresponding to VEGF165/189 (194 bp) is evident in human brain, bladder and kidney (AMS Biotechnology Ltd), and prostate and kidney (Primer Design Ltd) using forward and reverse primers designed to exon 7 (DB exon7a) and the 3'UTR (DB 3'UTR) respectively. No products corresponding to VEGF165b/189b (128 bp) are evident in any of these samples.

    Article Snippet: PCR reactions were routinely performed in 25 µl using 1 µl of cDNA (AMS cDNAs) and 20 µl using 5 µl of cDNA (Primer Design cDNAs).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Affinity Magnetic Separation

    Discriminating VEGF isoform-specific RT-PCR highlights the importance of primer design in influencing detection of putative VEGFxxxb products. RT-PCR was performed using an exon 3 forward primer together with different VEGFxxxb isoform specific reverse primers designed to detect VEGF188b and/or VEGF164b or VEGF120b. A , RT-PCR using a reverse primer spanning 13 bases across the exon8b/exon7 junction (164b/188b-2) amplified a putative VEGF164b (311 bp) product in wt, VEGF164 VEGF120-expressing tumours and a putative VEGF188b product (383 bp) in VEGF188-expressing tumours. B , More discriminating RT-PCR using a 164b/188b-3 reverse primer spanning only 4 bases across the exon8b/7 splice-junction failed to reveal VEGFxxxb PCR products. The ability to detect VEGFxxx products in reactions using exon8 and 3′UTR-C reverse primers highlights efficient PCR conditions. C , No products corresponding to VEGFxxxb isoforms were generated by RT-PCR using cDNA from mouse fibrosarcoma cells and a reverse primer spanning 5 bases across the exon8b/7 junction (164b/188b-1); VEGF188b (1209 bp) and VEGF164b (1137 bp) products were amplified from pPNT VEGF 164 (V 164 ) pPNT VEGF 188 (V 188 ) ‘knock-in’ plasmid vector cDNA templates [23] . D E , Putative VEGF188b and/or VEGF164b products were detected in RT-PCR reactions using reverse primers spanning 13 bases across the exon8b/7 junction (164b/188b-2 164b/188b-4) in fibrosarcoma cells and pPNT VEGF 164 pPNT VEGF 188 plasmids. F G , Products corresponding to VEGF120b (179 bp) species were readily detected in all fibrosarcoma tumour and cell extracts with a VEGF120-specific reverse primer spanning 16 bases across the exon8b/5 junction (120b-2), whilst no VEGF120b products were generated when the reverse primer spanned only 5 bases across the 8b/5 junction (120b-1).

    Journal: PLoS ONE

    Article Title: Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

    doi: 10.1371/journal.pone.0035231

    Figure Lengend Snippet: Discriminating VEGF isoform-specific RT-PCR highlights the importance of primer design in influencing detection of putative VEGFxxxb products. RT-PCR was performed using an exon 3 forward primer together with different VEGFxxxb isoform specific reverse primers designed to detect VEGF188b and/or VEGF164b or VEGF120b. A , RT-PCR using a reverse primer spanning 13 bases across the exon8b/exon7 junction (164b/188b-2) amplified a putative VEGF164b (311 bp) product in wt, VEGF164 VEGF120-expressing tumours and a putative VEGF188b product (383 bp) in VEGF188-expressing tumours. B , More discriminating RT-PCR using a 164b/188b-3 reverse primer spanning only 4 bases across the exon8b/7 splice-junction failed to reveal VEGFxxxb PCR products. The ability to detect VEGFxxx products in reactions using exon8 and 3′UTR-C reverse primers highlights efficient PCR conditions. C , No products corresponding to VEGFxxxb isoforms were generated by RT-PCR using cDNA from mouse fibrosarcoma cells and a reverse primer spanning 5 bases across the exon8b/7 junction (164b/188b-1); VEGF188b (1209 bp) and VEGF164b (1137 bp) products were amplified from pPNT VEGF 164 (V 164 ) pPNT VEGF 188 (V 188 ) ‘knock-in’ plasmid vector cDNA templates [23] . D E , Putative VEGF188b and/or VEGF164b products were detected in RT-PCR reactions using reverse primers spanning 13 bases across the exon8b/7 junction (164b/188b-2 164b/188b-4) in fibrosarcoma cells and pPNT VEGF 164 pPNT VEGF 188 plasmids. F G , Products corresponding to VEGF120b (179 bp) species were readily detected in all fibrosarcoma tumour and cell extracts with a VEGF120-specific reverse primer spanning 16 bases across the exon8b/5 junction (120b-2), whilst no VEGF120b products were generated when the reverse primer spanned only 5 bases across the 8b/5 junction (120b-1).

    Article Snippet: PCR reactions were routinely performed in 25 µl using 1 µl of cDNA (AMS cDNAs) and 20 µl using 5 µl of cDNA (Primer Design cDNAs).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Polymerase Chain Reaction, Generated, Knock-In, Plasmid Preparation

    Effect of IGF-1 and TGFβ-1 on expression of VEGF isoforms in mouse podocytes. Cell lysates were prepared from untreated control (C) podocyte cells or cells treated with either 100 nM IGF-1 (IGF) or 1 nM TGFβ-1 (TGF) for 12 hours in serum free media. Results from three independent experiments are shown. RT-PCR using general primers designed to simultaneously amplify both VEGFxxx and VEGFxxxb isoforms (exon7a/3'UTR) revealed only VEGFxxx (194 bp). Qualitative assessment of the PCR products suggests that treatment with either growth factor increased VEGFxxx expression. The same RT-PCR strategy using three different extracts (1, 2, 3) from HEK 293 cells similarly revealed only VEGFxxx (194 bp). (-tem) corresponds to reactions containing water instead of cDNA. (-RT) corresponds to reactions containing water instead of reverse transcriptase.

    Journal: PLoS ONE

    Article Title: Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

    doi: 10.1371/journal.pone.0035231

    Figure Lengend Snippet: Effect of IGF-1 and TGFβ-1 on expression of VEGF isoforms in mouse podocytes. Cell lysates were prepared from untreated control (C) podocyte cells or cells treated with either 100 nM IGF-1 (IGF) or 1 nM TGFβ-1 (TGF) for 12 hours in serum free media. Results from three independent experiments are shown. RT-PCR using general primers designed to simultaneously amplify both VEGFxxx and VEGFxxxb isoforms (exon7a/3'UTR) revealed only VEGFxxx (194 bp). Qualitative assessment of the PCR products suggests that treatment with either growth factor increased VEGFxxx expression. The same RT-PCR strategy using three different extracts (1, 2, 3) from HEK 293 cells similarly revealed only VEGFxxx (194 bp). (-tem) corresponds to reactions containing water instead of cDNA. (-RT) corresponds to reactions containing water instead of reverse transcriptase.

    Article Snippet: PCR reactions were routinely performed in 25 µl using 1 µl of cDNA (AMS cDNAs) and 20 µl using 5 µl of cDNA (Primer Design cDNAs).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Transmission Electron Microscopy

    Generation of recq2 null mutants in bloodstream form T. brucei . ( A ) PCR was used to amplify a 5’ and a 3’ region (blue arrows) of the RECQ2 ORF (orange arrow). These PCR products were the ‘targeting regions’ cloned into the constructs shown below,which weremade to generate heterozygous (+/-) and knockout (-/-) mutants by replacing the ORF with resistance cassettes. Generalised representation of the linearised blasticidin and neomycin (G418) resistance constructs relative to the RECQ2 ORF (orange) and the targeting flanking regions (UTR; grey), which allow HR-mediated exchange (crosses). β/α tub, β/ α tubulin intergenic region; actin IR, actin intergenic region; BSD , blasticidin resistance; NEO , neomycin (G418) resistance. Not to scale. ( B ) The upper diagram shows diagnostic PCRs to confirm replacement of RECQ2 with the knockout constructs (positions of primers are indicted by arrows): a region of the ORF was amplified (“ORF PCR”) and, in addition, a region was amplified using a forward primer lying upstream of the 5’ UTR region in the knockout construct and a reverse primer specific to the BSD or NEO genes (“BSD PCR” and “NEO PCR”, respectively). The lower diagram shows agarose gels of PCR products generated from genomic DNA from wild type (WT) cells and heterozygote (+/-) and knockout (-/-) clones using primers described above. Distilled water was used as a negative control (-). Broken lines indicate different images aligned in this figure; size markers are shown (bp). ( C ). RT-PCR confirmation of recq2 mutants. A region of the PIF6 ORF and a region of the RECQ2 ORF was PCR-amplified using cDNA (+RT) synthesised from RNA extracted from wild type (wt) cells and from recq2 +/- and recq2 -/- mutants; control samples in which no reverse transcriptase had been added (-RT) are shown, as are controls using distilled water rather than substrate (-); size markers on the agarose gels are shown (bp). DOI: http://dx.doi.org/10.7554/eLife.12765.004

    Journal: eLife

    Article Title: Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

    doi: 10.7554/eLife.12765

    Figure Lengend Snippet: Generation of recq2 null mutants in bloodstream form T. brucei . ( A ) PCR was used to amplify a 5’ and a 3’ region (blue arrows) of the RECQ2 ORF (orange arrow). These PCR products were the ‘targeting regions’ cloned into the constructs shown below,which weremade to generate heterozygous (+/-) and knockout (-/-) mutants by replacing the ORF with resistance cassettes. Generalised representation of the linearised blasticidin and neomycin (G418) resistance constructs relative to the RECQ2 ORF (orange) and the targeting flanking regions (UTR; grey), which allow HR-mediated exchange (crosses). β/α tub, β/ α tubulin intergenic region; actin IR, actin intergenic region; BSD , blasticidin resistance; NEO , neomycin (G418) resistance. Not to scale. ( B ) The upper diagram shows diagnostic PCRs to confirm replacement of RECQ2 with the knockout constructs (positions of primers are indicted by arrows): a region of the ORF was amplified (“ORF PCR”) and, in addition, a region was amplified using a forward primer lying upstream of the 5’ UTR region in the knockout construct and a reverse primer specific to the BSD or NEO genes (“BSD PCR” and “NEO PCR”, respectively). The lower diagram shows agarose gels of PCR products generated from genomic DNA from wild type (WT) cells and heterozygote (+/-) and knockout (-/-) clones using primers described above. Distilled water was used as a negative control (-). Broken lines indicate different images aligned in this figure; size markers are shown (bp). ( C ). RT-PCR confirmation of recq2 mutants. A region of the PIF6 ORF and a region of the RECQ2 ORF was PCR-amplified using cDNA (+RT) synthesised from RNA extracted from wild type (wt) cells and from recq2 +/- and recq2 -/- mutants; control samples in which no reverse transcriptase had been added (-RT) are shown, as are controls using distilled water rather than substrate (-); size markers on the agarose gels are shown (bp). DOI: http://dx.doi.org/10.7554/eLife.12765.004

    Article Snippet: RNA was extracted from the cells using the Qiagen RNeasy kit, and cDNA synthesis was performed using random primers and the Primer Design Precision nanoScript Reverse Transcription kit (Primer Design), according to manufacturer’s instructions.

    Techniques: Polymerase Chain Reaction, Clone Assay, Construct, Knock-Out, Diagnostic Assay, Amplification, Generated, Negative Control, Reverse Transcription Polymerase Chain Reaction

    Generation of TbRECQ2 mutants in T. brucei HR1 and HRES cells. ( A ) Agarose gels of PCR products generated from genomic DNA from HRES or HR1 wild type (WT) cells and RECQ2 heterozygote (+/-) and knockout (-/-) clones using primers described in the Figure S1B . Gaps indicate that lanes have been aligned in this figure after excision from multiple gels or from disparate parts of the same gel; size markers are shown (bp). ( B ) Confirmation by PCR of RECQ2 knockout in HR1 and HRES cell lines. Agarose gels showing products generated when PCR was performed on a region of the PIF6 ORF or the RECQ2 ORF using cDNA (+RT) synthesised from RNA extracted from recq2 -/- mutants; control reactions in which no substrate was added (-), or where cDNA from HR1 WT cells was used, are shown. Gaps indicate that lanes have been aligned in this figure after excision from multiple gels or from disparate parts of the same gel; size markers are shown (bp). DOI: http://dx.doi.org/10.7554/eLife.12765.007

    Journal: eLife

    Article Title: Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

    doi: 10.7554/eLife.12765

    Figure Lengend Snippet: Generation of TbRECQ2 mutants in T. brucei HR1 and HRES cells. ( A ) Agarose gels of PCR products generated from genomic DNA from HRES or HR1 wild type (WT) cells and RECQ2 heterozygote (+/-) and knockout (-/-) clones using primers described in the Figure S1B . Gaps indicate that lanes have been aligned in this figure after excision from multiple gels or from disparate parts of the same gel; size markers are shown (bp). ( B ) Confirmation by PCR of RECQ2 knockout in HR1 and HRES cell lines. Agarose gels showing products generated when PCR was performed on a region of the PIF6 ORF or the RECQ2 ORF using cDNA (+RT) synthesised from RNA extracted from recq2 -/- mutants; control reactions in which no substrate was added (-), or where cDNA from HR1 WT cells was used, are shown. Gaps indicate that lanes have been aligned in this figure after excision from multiple gels or from disparate parts of the same gel; size markers are shown (bp). DOI: http://dx.doi.org/10.7554/eLife.12765.007

    Article Snippet: RNA was extracted from the cells using the Qiagen RNeasy kit, and cDNA synthesis was performed using random primers and the Primer Design Precision nanoScript Reverse Transcription kit (Primer Design), according to manufacturer’s instructions.

    Techniques: Polymerase Chain Reaction, Generated, Knock-Out, Clone Assay