DNA sequencing facility | Genomics Facility

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DNA Sequencing

DNA sequencing facility

We currently provide capillary sequencing using Beckman CEQ 2000 XL [ http://www.beckman.com ]. The procedure how to prepare samples for sequencing can be found by clicking [Read more] below:

Samples for DNA sequencing

DNA template:
The template can be either a plasmid or a PCR fragment. It is important to prepare sufficient template to allow an accurate quantitation and purification.
Note! We can not guarantee a good result if you not give us a template which is not purified. This is specially important when you give us PCR products since the unpurified product contain unused primers and nucleotides.

Sequencing primer:
The primer should be properly purified, at least de-salted. Primers should be at least 18 bases long to ensure good hybridization. The best primers for cycle sequencing are between 23-26 bases in length. In addition, primers should have a melting temperature (or Tm) of greater than 55Â°C, and the optimum Tm is above 60Â°C for the thermal cycling conditions that we use. The GC content of primers should be around 50%. Again, if this is not possible, a longer primer should be designed to bring the Tm up to the recommended range.

Preparing sequencing samples:
Prepare your samples in ddH2O containing 10 Âµl of a mix of both the template and the primer. This mix should contain 200 fmole of template and 10 pmole of the primer. It is important that the mix doesnâ€™t contain traces of EDTA. The water shall NOT be DEPC treated.
You should use strips of 8 tubes and label them with your initials and a number. Contact Karin Degerman the first time you leave sequencing samples so she will know to whom the samples belong. Put the samples in the freezer opposite Roland Wass office on floor 3.

Formulas:
For dsDNA: 200 fmole template = 130 x kilo basepairs (ng)

Example: plasmid 3000 bp
200 fmole equals 130 x 3,0 ng = 390 ng

For primers: 1ÂµM = 1 pmole/Âµl

Sequencing:
We run the sequencing reactions for you. We run the samples on our capillary electrophoresis system CEQ2000XL (Beckman), where we use the Dye terminator Cycle Sequencing kit form Beckman. The output file will be in .scf file format.

Fetching the samples:
Contact Karin Degerman and she will give you information of how to fetch your sequences from the ABI377 computer.

Freeware programs for opening your sequences:
Mac users:
4Peaks (will open both .abi and .scf formats) (MacOS X)
http://mekentosj.com/4peaks/

PC users: BioEdit
http://www.mbio.ncsu.edu/BioEdit/bioedit.html

July 2010

Friday, July 30, 2010
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