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Sanger Sequencing 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.

  • Reda_smallPlasmid DNA should be supplied in 10µl water at a concentration of 100ng/µl per sequencing reaction
  • Cosmid DNA should be supplied in 10µl water at a concentration of 150ng/µl per sequencing reaction
  • PCR fragments should be supplied in 10µl water at a concentration of 20ng per 100 base pairs

PCR product:

- 100 to 200 bp  supply 1 to 3 ng/ul

- 200 to 500 bp supply 3 to 10 ng/ul

- 500 to 1,000 bp supply 5 to 20 ng/ul

- 1,000 to 2,000 bp supply 10 to 40 ng/ul

- >2,000 bp supply 40 to 100 ng/ul

Any non-standard primers submitted should be at a concentration of 10pm/µl (10µM) in water. We use 2µl 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 and 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
Old Addenbrookes Site

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

Results are sent via email so please ensure that the mailbox size is sufficient.

Our Fragment Analysis Service is now provided on the ABI 3130XL. Please contact the Facility for further information.