I am doing cloning now by using infusion cloning method, but I am not getting good primers. For infusion primer, we have to add 15bp homologous to 5'. When I get a specific primer, then it has dimer and hairpin problems, but when there is no dimer and hairpin structure problem, I lose specificity. Can anyone suggest me how can I solve this problem?
For cloning purpose, unspecific is not that hard to get rid of.
1. using the first pair of primers, with 5 or 10 time amount of regular concentration. And with correct annealing temperature, there should be enough product.
2. using the second pair of primer, after PCR, run a high conc. of gel (like 3%) for a long time, then cut gel for isolation the correct amplicon.
3. if possible, redesign primers at outer sides of your gene.
1. About the hear pin structures:
Often you can get hear pin (HP) structures important to know is: at which temperature those structures will melt. If your annealing temperature is higher than the melting temperature of the HP structure you are OK.
Example: your Tm=60oC
HP=55oC
If you set up your PCR annealing at 57-58oC you will be OK because the HP will melt at 55oC
2. About the primer dimers (self dimer or heterodimers) if those dimers are not more than 20% you will still be OK
3. tricks when you have primer dimers:
>use more of the For or more of the Rev primer (the one predicted to make dimmers)
>if you have heterodimer set up few cycles (2-5) with one primer only (the one that is more problematic).
Than pause the machine and add the second primer and continue run for ~30 more cycles.
4. When designing primers for infusion try to have GC=50% or more.
If bellow > use longer overlap (20-25bp) to compensate
I do not know what software you use for analysis of your primers> Try IDT DNA oligoanalizer
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
If you need further help let me know
Thank you Fang Liang and Zoia Stoytcheva for your valuable suggestions.
Sincerely,
Sailesh
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