Protocol Online logo
Top : Forum Archives: : Molecular Biology

Primer design help - secondary structures - Please help me troubleshoot? (May/13/2008 )

Pages: Previous 1 2 

QUOTE (MolBioGirl @ May 15 2008, 12:57 PM)
I usually design ~22-25nt primers, with sequences a little more GC rich on the 5' end and AT rich on the 3' end. I use software (Primer Select or VectorNTI) to check for hairpins and dimers. I try to avoid any hairpin with a negative delta G, and dimers with anything below ~ -3 delta G. (Redesign if needed!) My TM's can range anywhere from ~60°C - ~75° or 80°, depending what I'm working on. But I try to make sure the primers are within 5° of each other, and usually set my annealing temp 5-10° below my lowest TM.

Thanks for the info... however if I HAVE to design primers that corresponds to a certain region of the gene that just means that I don’t have much of a choice, true? For example the sequence I am interested in may have a gigantic stretch of G/T but not so much A/T…

It’s not quite like designing sequencing primers where I can be flexible with which DNA stretch I choose just as long as it’s evenly spaced out. True?


True, but sometimes you can choose a new primer sequence that's shifted just a little (on your flanking region) from where you originally were -- and it can make a big difference. Also, sometimes you can change just one or two nt's in the primer sequence to disrupt a hairpin or dimer (depending what your goal is though, you may not be able to)... Also, you should avoid stretches of more than 3 of any single nt to reduce mispriming. (if you can help it, especially G or C). GC rich sequences can be hard to amplify. If you have a lot of trouble, try adding 5% DMSO.
Hope this helps smile.gif


I need a same kind of help.
I am designing primers to amplify a 10 kb region, (5kb upstream form Exon2 and 5kb downstream of exon 2), which needs addition of restriction sites to fwd and rew primers.
I have come up with primers as
5' - ATCG GGTACC ATG(start) .....complimentary bases
3' - AAGCT GCGGCCGC TTA(stop)...
Do they look ok. My question was, do I need to incorporate start and stop codon ATG and TTA to the primer, or find a site like say NNNNATG in the mRNA of mine.And then design the primer from there on. Also does the clamp sequence goes first, then RE site and then the ATG for fwd primer. Similarly does the rew primer needs to be cap seq, RE and then TTA???
I am using the vector pcDNA as it has the same RE sites of Acc65I and NotI in the MCS region.
This is the first time I am designing primers. So any help or advice will be helpful.


Pages: Previous 1 2