Sanger Sequencing


The TCGA performs Sanger sequencing on PCR products, plasmids, single stranded DNA, double stranded DNA, cosmids, BAC DNA and Bacterial genomic DNA using an Applied Biosystems 3500xL Genetic Analyser. Sanger sequencing (or DNA sequencing by capillary electrophoresis) remains an informative, quick and cost effective tool for use in life science research. It can be performed with user-supplied primers or any of the TCGA’s standard sequencing primers. Templates for sequencing can be supplied in individual tubes or 96 well plates.

Template/primer concentration guide

Template Template concentration Minimum volume required Primer concentration (3 µl per reaction)
Plasmids 150-500ng/µl 5 µl 3.2 pmol/µl
PCR products
100-200 bp 1-3 ng/µl 5 µl 3.2 pmol/µl
200-500 bp 3-10 ng/µl 5 µl 3.2 pmol/µl
500-1000 bp 5-20 ng/µl 5 µl 3.2 pmol/µl
1000-2000 bp 10-40 ng/µl 5 µl 3.2 pmol/µl
>2000 bp 20-50 ng/µl 5 µl 3.2 pmol/µl
Single stranded DNA 25-50 ng/µl 5 µl 3.2 pmol/µl
Cosmid, BAC DNA 0.5-1.0 µg/µl 5 µl 3.2 pmol/µl
Bacterial genomic DNA 2-3 µg/ul 5 µl 3.2 pmol/µl

Lower template concentrations may be analysed, please discuss with the TCGA.

PCR products should be purified to remove unincorporated dNTPs and primers; we recommend Illustra ExoProStar™ 1-step (Fisher Scientific catalogue number #12124082) or ExoSAP-IT™ PCR Product Cleanup (Thermo Fisher #78250.40.UL).

DNA Sequencing Request Form

 

Standard Sequencing Primers

The following standard sequencing primers are available through the TCGA:

Name Sequence (5` to 3`) Length
M13 rev (-29) CAG GAA ACA GCT ATG ACC 18bp
M13 rev (-49) GAG CGG ATA ACA ATT TCA CAC AGG 24bp
M13 uni (-21) TGT AAA ACG ACG GCC AGT 18bp
SP6 CAT TTA GGT GAC ACT ATA G 19bp
T3 AAT TAA CCC TCA CTA AAG GG 20bp
T7 TAA TAC GAC TCA CTA TAG GG 20bp
T7 term CTA GTT ATT GCT CAG CGG T 19bp
pCR 3.1 (BGHrev.) TAG AAG GCA CAG TCG AGG 18bp
pGex for ATA GCA TGG CCT TTG CAG G 19bp
pGex rev GAG CTG CAT GTG TCA GAG G 19bp
CMV-for CGC AAA TGG GCG GTA GGC CTG 21bp
CMV-rev CCT CCA CCC CAT AAT ATT ATA GAA GGA CAC 30bp
U19 GGT TTT CCC AGT CAC GAC G 19bp

 

Please contact us with any questions or requests.