5 replies to this topic

[Shiny]

Hello everyone,

I am trying to insert an epitope tag at the end of my protein and I want to use PCR to clone the fragment in. Now the problem is the reverse primer is very longer as it incorporates the tag (nearly 100 nucleotides) in comparison to forward primer (20 nucleotides). For the first round the reverse primer anneals to the 20 odd nucleotides of the template DNA while the tag sequence is in the tail of the PCR, however for the subsequent rounds the reverse primer now is as long as 100 nucleotides which is no match for a 20bp forward primer.

Has anyone encountered a similar problem. Any information would be useful

thanks

Shiny

[GNANA]

i hav'nt encountered such prob, at the same time i wouldnt have chosen to insert the tag this way...rather i would have ligated the tag seperately either into the PCR product or into the vector ....

[bob1]

Don't worry about the annealing temperature for the tag at all, your T_A should be based on the specific primer only.  You only need the first round to work, and then it will be fine.

[Shiny]

but what about the subsequent cycles of PCR for amplifying the product, then the TA of the primers will be a complete mismatch!!

[Shiny]

Okay so as suggested I set up a PCR with the annealing temperature of the primer pair and I do not have any PCR product. Besides that I also tried to set up a PCR individually with the two primers and mix them together and run a normal PCR reaction however I do not see any PCR products. Please help any suggestions would be helpful.

[Trof]

Too long primer may have other problems. With such length it will likely create more "binding domains" that would behave as separate primers. Or it may form a secondary structure that will prevent binding at all.
Maybe I would try to play with DMSO.