Hi all!
I want to sequence a 4.6 kb region from a 5.1 kb purified PCR product (PCR product includes neighbouring regions of region of interest). I'll be sending pre-mixed reactions to GeneWiz.
I understand I will have to design several primers for several reactions to cover the entire length of the region. How far appart should each forward primer be to the next one? How much overlap should I include between each forward and reverse primer?
I've designed a tentative strategy which consists of 5F and 5R primers. There are 800 bp between each successive F primers and between each successive R primers. There's 200 bp between each F and R primers (to account for unreliable reads at the beginning of each reaction). Does this make sense? Could I lengthen the distance between the primers to have less reactions? 10 reactions means I would need 4.6 ug DNA, which is a lot to generate and purify. Is there a better way to go about this?
Thanks so much!
I think that your total amount of template needed is pessimistic.
Usually we use 25ng x length of template in kb for each sequencing reaction so 5.1 x 25 or about 125ng for each reaction.
I would not worry too much about the quality of the primers....there is only 3.6kb ( a very small genome ) to anneal to so most sequences will work and we do not need to worry about heterodimers because there is only one sequencing primer. I expect to get 800bases of good sequence so if the first sequence is 0-800 bases design a second forward primer at 700 then 700-720 is primer so no sequence and the next 30 - bases are poor quality then you get at least 30 well sequenced overlap bases in the forward direction and continue with overlapping 700 bases up to the end of the sequence. It looks like 7 forwards primers and 8 reverse primers if you are sequencing in both directions and about 1450ng of template needed 700ng if sequencing only in one direction
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