Sanger Sequencing Sample Preparation and Submission

 

  • TAGC will only process samples pre-mixed by client. The primers listed below can be purchased from TAGC for $6/100 uL aliquot.

 

  • TAGC will do Sanger sequencing on Tuesday, Thursday and Friday only. Samples will be accepted every week day.

 

 Effective July 15, 2015:

  • The deadline for sample drop off is 3:30 pm the day before a Sanger sequencing day

 

Effective October 5, 2015:

  • TAGC will accept Sanger sequencing samples in strip tubes format only
  • Strip tubes are available in TAGC and the cost is included in the sequencing price 

 

 

We sequence PCR products, plasmids, cosmids, BACs and bacterial genomic DNA.

Each sequencing reaction contains the template DNA (10 uL), the primer (1 uL) and the dNTPs/ddNTPs and DNA polymerase mix (9 uL).

For GC-rich templates we add DMSO to the reaction mix to a final concentration of 5%. If sequencing GC-rich templates, please specify on the Sanger Sequencing Form.

 

 

  • Template DNA

It is recommended that the template be diluted in laboratory-grade water or Tris-HCl, pH 7.5, not TE. Buffers containing EDTA cause inhibition of sequencing reactions by chelating the co-factors of DNA polymerase. Qiagen elution buffer, EB, is acceptable as it does not contain EDTA.

PCR templates must be cleaned-up to remove unincorporated nucleotides and primers. Any traces of primers from the PCR will result in a double sequence. Any remaining dNTPs and salts will alter the concentration of similar components in the sequencing kit, with unfavourable results. For more information on the PCR clean-up protocols please contact TAGC.

The purity and the concentration of the DNA template are critical for the quality of the sequencing results. Please ensure that the template DNA is free of impurities, such as salts, ethanol, phenol/chloroform, RNA and proteins and it is within the recommended concentration range.

If the DNA template is too concentrated, the sequencing reaction could drop off quickly, significantly reducing the read length.

If the template concentration is too low, the relatively high background noise will introduce errors into the sequence analysis.

 

Template DNA

Size

Concentration

PCR

100-200 bp

0.1-0.3 ng/uL

200-500 bp

0.3-1 ng/uL

500-1,000 bp

0.5-2 ng/uL

1,000-2,000 bp

1-4 ng/uL

>2,0000 bp

2-5 ng/uL

Single stranded DNA

 

2.5-5 ng/uL

Plasmid

 

15-22.5 ng/uL

Cosmid, BAC

 

50-100 ng/uL

Bacterial genomic

 

200-300 ng/uL

 

  • Primer

The following commonly used primers can be purchased from TAGC for $6/100 uL aliquot. The primer aliquots are already diluted at the required concentration 3.2 pm/uL (3.2 micromolar) and ready to use.

  • M13-Fwd:  5′-TGT AAA ACG ACG GCC AGT-3′
  • M13-Rev:  5′-CAG GAA ACA GCT ATG AC-3′
  • T7-Fwd:  5′-TAA TAC GAC TCA CTA TAG G-3′
  • T7-Rev:  5′-GCT AGT TAT TGC TCA GCG G-3′
  • T3:  5′-AAT TAA CCC TCA CTA AAG GG-3′
  • TKpA:  5′-CTT CCG TGT TTC AGT TAG C-3′
  • SP6:  5′-GAT TTA GGT GAC ACT ATA G-3′
  • BGH-R:  5′-TAG AAG GCA CAG TCG AGG-3′
  • CMV:  5′-CGC AAA TGG GCG GTA GGC GTG-3′
  • pGEX3:  5′-CGG GAG CTG CAT GTG TCA GAG G-3′
  • pGEX5:  5′-GGG CTG GCA AGC CAC GTT TGG TG-3′
  • pJet1.2F:  5′-CGA CTC ACT ATA GGG AGA GCG GC-3′
  • pJet1.2R:  5′-AAG AAC ATC GAT TTT CCA TGG CAG-3′

 

It is recommended that the primer be diluted to a concentration of 3.2 pm/uL (3.2 micromolar) in laboratory-grade water or Tris-HCl, pH 7.5, not TE.

 

  • Pre-mix template DNA and primer

In a PCR tube, mix 10 uL template and 1 uL primer per reaction. Strips of 8 PCR tubes with caps attached are available in TAGC for your convenience. The cost of the strip tubes is included in the sequencing price.

Please label the cap with the corresponding sample number from the Sanger Sequencing Form and the side of the first and the last tube in the strip with your initials.

When sequencing large plasmids (>8 Kb) or bacterial genomic DNA, mix 10 uL template and 3 uL primer per reaction.

If sequencing the same template with multiple primers, prepare individual tubes for each pair of template – primer. Never mix more than one primer with the template.

 

 

Sample drop off: 7-121 Katz Building, 9 am – 12 pm and 1 pm – 3:30 pm.

Please submit your Sanger sequencing request through iLAB. More info at  https://www.med.ualberta.ca/research/corefacilities

 

 

 

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