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. The annealing temperature should be at least 50°C (see PCR program below).
Preparing sequencing samples:
Prepare your samples in ddH2O containing 15 µl of a mix of both the template and the primer. This mix should contain 300 fmole of template and 15 pmole of the primer. It is important that the mix doesn’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: 300 fmole template = 195 x kilo basepairs (ng)
Example: plasmid bp
300 fmole equals 195 x 3,0 ng = 585 ng
For primers: 1µM = 1 pmole/µl
Sequencing:
We run the sequencing reactions for you. Routinely we use the DYEnamic ET terminator cycle sequencing kit from ABP. The cycle sequencing reaction program is as follows:
95°C, 20 s (denaturation)
50°C, 15 s (annealing)
60°C, 1 min (extension)
28 cycles
Fetching the samples:
Contact and she will give you information of how to fetch your sequences from the ABI377 computer.
Freeware programs for sequence analysis:
Mac users: EditView 1.0.1 (not a full analysis program)
http://www.appliedbiosystems.com/support/software/
PC users: BioEdit
http://www.mbio.ncsu.edu/BioEdit/bioedit.html#downloads
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