so lost! stupid quenstion about primers.... - designing the primers (Nov/18/2005 )
i have designed the primers containing the RE sites and some clamp bases on each site....but i think i did it all wrong!
i think there is something about primers which i dont get....
if I have a sequence lets say:
5`-ACC ATG ATG CTG-3`
3`-TGG TAC TAC GAC-5`
if to suppose that each primer has three letters then...]
sense primer should be : 5`-TGG-3`
and anti-sense primer should be: 5`-GTC-3`
am I wrong? please helpme to understand this...
another question in the primer i insert the RE site as it is or a complementary sequnce...please give example if you have time....thanx a lot
Your primer should always be 5' to 3'. If you thinks about it, you'll realize your primer will always be the beginning of your synthesized strand, thus its sequence should match the beginning of the strand to be synthesized, and be complementary to the other strand. Given the following sequence, and assuming I was going to prime with six-mers:
5`-ACC ATG CTC GAC ATG CTG-3`
3`-TGG TAC GAG CTG TAC GAC-5`
My forward primer would be 5'-ACCATG-3', and my reverse primer would be 5'-CAGCAT-3'.
If the RE site you're adding is a palindrome, just tack them on the 5' end following a few irrelevant bases to allow the RE to cut. For example, if I wanted to add BamHI sites to the primers above, I would order:
F_primer: GTAC GGATCC ACCATG
R_primer: TACG GGATCC CAGCAT
The four bases before the BamHI site are there to allow the enzyme to cut effectively, what bases they are is not important. I routinely take the last four bases of my primer's sequence in reverse order (as I've done above), if nothing else, this will stop the ends from base pairing to each other...
BTW, while it is essential for you to understand all this stuff, I usually leave the actual primer picking to a program like Primer3 (see here). This program generates your primers in the correct 5'-to-3' direction for ordering, and watches out for many other primer defects (Tm differences, self complementarity, 3' stability, poly-X runs, %GC, etc.) -- any good primer picking program will do this for you. I also find it convenient to cut and paste my sequence in, and cut-and-paste my primers out, and right on to the order (I order mine via the web), thus there is no opportunity for me to introduce a typographical error...
Hope this helps!
thank you so much ....i just have to think of primers as a new strand to be synthesized.... have a great day !