Skip to main content
Apply

Ferguson College of Agriculture

Plasmid Sequencing Template Requirements

Optimum sequence quality depends on the quality if the template. Plasmid preps should be free of solvents and contamination. Plasmids should migrate at the predicted size and produce the expected band sizes upon digestion. See table below for information on required concentrations and necessary volumes.

PlasmidConcentration (ng/uL)Volume per reaction
Standard Service Sequencing50 ng/uL10 uL
Full Service Sequencing> 25 ng/uL10 uL

PCR Sequencing Template Requirements

Optimum sequence quality depends on the quality if the template. PCR preps should be free of solvents and contamination. PCR bands should migrate at the predicted size and not display evidence of an secondary bands. See table below for information on required concentrations and necessary volumes.

PCRConcentration (ng/uL)Volume per reaction
Standard Service Sequencing15 ng/uL2 uL per 250 bases
Full Service Sequencing> 2.5 ng/uL2 uL per 250 bases

Primers for DNA Sequencing

The Core Facility provides a selection ofcommon primersfor use in sequencing reaction without additional charge.

For many sequencing reactions applications, customers will provide primers specific for their sequences. Customers providing their own primers should have a primer aliquot in a separate tube alongside their sample (Don't mix the primer with the sample).

Primers should be provided at a concentration of 5 uM (5 pmol/uL = 5 uM = 1:20 dilution of 100 uM primer stock). We require at least 3 uL of primer per reaction.

House Primers

We provide most common sequencing primers (see below). These primers must be requested on the ‘Primer’ section of the sample submission form.

House Primers
T3ATT AAC CCT CAC TAA AG
T7 terminatorGCT AGT TAT TGC TCA GCG
T7 promoter - LongTAA TAC GAC TCA CTA TAG GG
T7 promoterAAT ACG ACT CAC TAT AG
M13 universalGTA AAA CGA CGG CCA GT
M13 reverseAAC AGC TAT GAC CAT GA
SP6GAT TTA GGT GAC ACT ATA G
S-TagCGA ACG CCA GCA CAT GGA C
malEGGT CGT CAG ACT GTC GAT GAA GCC
BGH reverseTAG AAG GCA CAG TCG AGG C
M13R(-48)AGC GGA TAA CAA TTT CAC ACA GG
pBad3GAT TTA ATC TGT ATC AGG C
KSTCG AGG TCG ACG GTA TC
SKCGC TCT AGA ACT AGT GGA TC
M13R (-29)CAGGAAACAGCTATGAC