design primers - (Apr/04/2008 )

I'm uisng gateway technology (by invitrogen) for my cloning. I have to design primers and have the follwing questions:

1) According to the manual, a given adapter sequence has to be added to my designed primer. So,

Given adaptor sequence (17bp) + 32bp given sequene (with shine dalgarno, kozac seqeunce, and ATG) + my selected primer secquence of 20bp (the manual says to add between 18-24bp and I have aded 20bp)= a primer with 69 nucleotides?!

2) Do my designed pimers have to be of the same length?

Thanks
Sarwat

The answer is Yes and Yes.
Your design primer is 20bp long.... That will determine the specificity of your amplification. So both forward and reverse designed primer should be same length.
However, you need to add adaptor sequence as well as ribosomal binding site to your forward primer. So you will end up with Forward primer 69 bp and reverse primer 20 bp.

This kind of amplification is not as efficient as normal PCR but is possible to be done.

Thank you for your reply. I should clarify my question further whn I ask if teh primers should be of teh same length.

According to the manual, a given adapter sequence has to be added to my designed primer. So,

forward primer: Given adaptor sequence (17bp) + 32bp given sequene (with shine dalgarno, kozac seqeunce, and ATG) + my selected primer secquence of 20bp (the manual says to add between 18-24bp and I have aded 20bp)= a primer with 69 nucleotides?!

reverse primer: Give adaptor seqeucen (17bp) + 13bp given sequence+ my selected primer secquence of 20bp= 50 nucleoides

I'm adding a constant 20bp of gene specific sequene to my forward and reverse primers. However, there are diferent lengths of given sequence from the manula to the F and R primers making them of diferent lengths?

Is ok. Altough your primers are long, but since you are using only a pair of primer... it should be fine.

Have you tried it out yet?
How is the difference of Tm between both of your primer?
How long your PCR product is?