I'm designing cloning primers for a fusion sequence. I need the whole sequence from the first nucleotide to the end. Unfortunately the beginning and the end are highly GC rich so the Tm is somehow above 70c and this is a problem for PCR efficiency!
Can anyone suggest any solution? Thanks
If you already have some, is more than enough for successful cloning it in any (cheap) PCR cloning kit compatible with your polymerase. With cloned insert you can play to subclone it in destination vector. An alternative is to run the PCR on this (kit-derived) plasmid, where all the sequence of your primers is already in. In this case I suggest to take 0.01-1ng of the template (no more) and run the PCR without the annealing step since the temperature of elongation is already the temp. of annealing...
Raheleh, use Primer3Plus to design your primers.
Nevertheless, even if they have a Tm of 80, you can always use 72 as annealing temperature. Lower temperature than primers' Tm won't hurt, but higher will be restrictive.
Also, how long do you want your primers to be? If the sequences are GC rich, then you either won't need long primers or they'll have high Tm.
use an enzyme capable of two step PCR- Accumprime from invitrogen for example. however, i would be surprised that those primers have trouble annealing at 55-60C- if you are anticipating a problem and not describing one you have- i will look to hear about your success and not your failure to clone this sequence.
thank you sezer,but in this case an additional sequence will be added to the target sequence and this would make problem because I want generate antibodies against this sequence.what should I do then?
Thank you Lisa,but the efficiency of primer annealing to the target sequence would decrease using lower tempratures than the primer needed temprature and I need high PCR efficiency
Suggest to show the sequence and over-hangs you want to add. What will be your template? cDNA or genomic one? With modern polymerases you don't have the problem with efficiency. For the antigen production you don't need the whole protein. You can move your primers just adding ATG if you truncate the N-terminus.
Give us know how the cloning is going, there are still many tricks...
Keep fingers cross:)
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