Sample Requirements
New! For information on our Next-Gen DNA sequencing service go to our 454 Sequencing page.
For information on our Cosmid DNA sequencing service go to our Cosmid Sequencing page.
For large sequencing orders we now offer a DNA preparation service. Please contact John Lester for further information.
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, the most commonly used type being the Tip 20. Traditional methods, ie. alkaline lysis, can work if care is taken.
For plasmids we require 10µl of DNA in water at a concentration of 100ng/µl per sequencing reaction.
Cosmids should be submitted at a concentration of 150ng/µl in water.
PCR fragments should be supplied at a concentration of 20ng per 100 base pairs in 10µl water.
Any non-standard primers submitted should be 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.
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 Pharmacia Gene Quant. This can give consistent accurate results and automatically provides abs. 260/280 ratio (which for best results should be around 1.8) however one must ensure careful use and that the cuvette used is very clean if spurious results are to be avoided.
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 and Cosmid 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
Cambridge
CB2 1GA
Sequencing requests from anywhere other than Biochemistry must be accompanied by a purchase order and a VAT exemption form where relevant.
Results are sent via email so please ensure that the mailbox size is sufficient. (N.B. only 12 results files can be sent to one email to a cam account, but there is no limit to mole addresses). Each sample generates a file, which is 280 kb in size. Sequence data can be viewed and edited using the following programs:
Older Macs use Editview
MacOSX use 4Peaks
Windows, Linux and MacOSX use FinchTV
|
