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Research Facilities at the Department of Biochemistry

 
Sanger sequencing 3730XL Applied Biosystems

Sequencer:

3730XL DNA Analyzer 

by Applied Biosystems from ThermoFisher is a 96-capillary array DNA Sequencer for traditional Sanger sequencing 

  • provides reads up to 1,000 bp
  • low cost per sample
  • typical 24 hour turnaround time

Sample Requirements

DNA submitted to the facility needs to be very pure, much purer for instance than for manual sequencing. It is for this reason that we recommend that DNA should be prepared using a commercial kit such as Qiagen. Traditional methods, ie. alkaline lysis, can work if care is taken.

 

  • Plasmid DNA should be supplied at a concentration of 100ng/µl and a volume of 3 µl per reaction* in water 
  • Cosmid DNA should be supplied at a concentration of 150ng/µl and a volume of 3 µl per reaction* in water 
  • PCR fragments should be supplied at a concentration of 20ng per 100 base pairs (see table below) and a volume of 3 µl per reaction* in water 

*Minimum 10 µl volume required to allow for evaporation or any other potential loss and any repeat reactions if necessary

PCR Product
Size Concentration
100-200 bp 1-3 ng/ul
200-500 bp 3-10 ng/ul
500-1,000 bp 5-20 ng/ul
1,000-2,000 bp 10-40 ng/ul
>2,000 bp 40-100 ng/ul

Any non-standard primers submitted should be at a concentration of 10pm/µl (10µM) in water. We use 3µl of primer solution per sequencing reaction, but please give us a minimum volume of 10µl to allow for evaporation or any other potential loss and any repeat reactions if necessary.

We need the correct amount of DNA as indicated above, not too little but also not too concentrated since we do not have the time to make further dilutions due to the large numbers of samples we receive. Most experienced sequencers will be able to make an accurate assessment of DNA quantity but some may have difficulty. One method is to use a Nanodrop. This can give consistent, accurate results and automatically provides abs. 260/280 ratio (which for best results should be around 1.8).

N.B. DNA for sequencing should always be supplied in water only and not TE or Tris buffer. Also please submit samples in tubes no smaller than 0.5ml to avoid handling problems.

Samples and completed DNA sequencing request forms can be dropped off in the basket provided in the Biochemistry reception (Sanger Building) or posted to the facility at the following address:

DNA Sequencing Facility
Department of Biochemistry
University of Cambridge
80 Tennis Court Road
Cambridge
CB2 1GA

Sequencing requests from anywhere other than Biochemistry must be accompanied by a printed purchase order.

 

Stock Primers

We supply some primers for use in sequencing reactions.

We also accept any custom primer. Please design your sequencing primers to be between 18 and 22 bases long. Each primer should have a GC content between 50 and 60%. The primer annealing temperature should be between 55 and 60°C.

Please supply your custom primers at a concentration of 10pm/µl (10µM) in water. We use 2µl of primer solution per reaction but please give us an excess to allow for evaporation or any other potential loss.

Stock Primers

Primer Sequence Length
M13 forward (-20F) 5'- TGT AAA ACG ACG GCC AGT - 3' 18
M13 reverse (-29R) 5' - CAG GAA ACA GCT ATG AC - 3' 17
T7 5' - TAA TAC GAC TCA CTA TAG GG - 3' 20
T7t 5' - GCT AGT TAT TGC TCA GCG G - 3' 19
Sp6 5' - CGA TTT AGG TGA CAC TAT AG - 3' 20
T3 5' - ATT AAC CCT CAC TAA AGG GA - 3' 20
pGEX 5' 5' - GGG CTG GCA AGC CAC GTT TGG TG - 3' 23
pGEX 3' 5' - CCG GGA GCT GCA TGT GTC AGA GG - 3' 23
-40F 5' - GTT TTC CCA GTC ACG AC - 3' 17
-47F 5' - CGC CAG GGT TTT CCC AGT CAC GAC - 3' 24
-48R (30nt_M13R) 5' - AGC GGA TAA CAA TTT CAC ACA GGA - 3' 24
-55R 5' - AAT TGT GAG CGG ATA ACA ATT TCA - 3' 24
BGH reverse 5' - TAG AAG GCA CAG TCG AGG - 3' 18
CMV forward 5' - CGC AAA TGG GCG GTA GGC GTG - 3' 21

 

Pricing

We offer a basic price of £5 per sequencing reaction for all our customers.

Large sequencing orders may qualify for a discount. Please contact the Facility for further details.

 

Turnaround times

We realise that turnaround time is extremely important to our customers and aspire to provide high quality results in the shortest possible time. Our current turnaround times are about 1 to 2 working days.