max primer length - 85 nt too long? - (May/27/2008 )

Hi,

did anybody try to use a Primer of a total length of 85 nt for PCR?
up to now I only tried it with max. 75 nt total length without having problems.

Into a short DNA sequence I want to introduce several features so I'd need to run 2 separate sets of PCR following by two separately done ligation / transformation steps what is quite time consuming.

I have done upto 94-mer PCRs relatively easily. The template had been BAC, so I can not be sure if you are doing genomic PCR or something. In all long-primer PCRs, I have found that as long as the priming sequence is short (20-30 bp), and the rest is just adapters and enzyme sites, it works well.

Hallo boz from neighour lab use primer long 106 bp, it works but on the 3 end he has lost 13bp after amplification. If you overcame internal palindroms repeats, it should principialy work
good luck

Any length is fine really, it's just the likelihood of an error in the primer will be higher so don't be surprised if that happens. You can get them purified by HPLC which i think which separates primers by size (by the base pair) so that if any of them have an insertion or deletion that primer can be removed. Bit more expensive but it's an option if you're worried. Go for the one long primer, it should be fine.

as killerkoz17 said, any size if find. I have done PCR with 100bp primers many times before.

My lab skips the HPLC purification as we have never seen the use for it.

Primers are built 3' to 5' with a capping step between addition of each nucleotide. The capping reaction is very efficient, though the addition of a new nucleotide is less so (>98.5%) Consequently at the end of the synthesis run, as much as 60% of the primers made are truncated. Sounds bad, but as the primers are built 3' to 5', all these truncated primers are missing the 5'end. If a restriction site is placed on the 5' end of the primer (to ligate into a plasmid) and you can dispose of the HPLC purification.

Also it is important to avoid internal palindromic sequence and minimise primer self annealing. Of course this is not always possible but due try. And if there are small internal palindromes and self annealing, giving the primer a high annealing temperature helps.

The annealing temperature of a large primer is calculated by considering only the part of the primer that binds to the template. The annealing temperatures I use for large primer is 60 Celsius, (no less than 58 C and as high as 62 Celsius)

A useful website to check your primer is Oligo calculator by Northwestern, it calculate Tm, and spots areas which self anneal or form hairpin loops