primer design - really necessary? (May/20/2009 )
Hi all
Now, I know that even for a simple PCR, primers have to be designed carafully.
I am trying to PCR a gene from genomic DNA of a bacteria (primers Tm is about 63 oC, annealing at 57 oC). After running a gel, however, so many non-specific bands present.
More interestingly, 
, I brought a new pair of primers with longer length (Tm is up to 73 oC, annealing at 67 oC), non-specific PCR is still there, but the band I need (at about 700bp) disappeared
Now, I am trying to design another primers. But how could I know whether my primers are specific enough to prevent non-specific annealing and non-specific PCR ??
Is there any program help you to calculate whether your primers will give non-specific PCR ??
ThX 
lactamase on May 20 2009, 03:44 AM said:
Now, I know that even for a simple PCR, primers have to be designed carafully.
I am trying to PCR a gene from genomic DNA of a bacteria (primers Tm is about 63 oC, annealing at 57 oC). After running a gel, however, so many non-specific bands present.
More interestingly,
, I brought a new pair of primers with longer length (Tm is up to 73 oC, annealing at 67 oC), non-specific PCR is still there, but the band I need (at about 700bp) disappeared Now, I am trying to design another primers. But how could I know whether my primers are specific enough to prevent non-specific annealing and non-specific PCR ??
Is there any program help you to calculate whether your primers will give non-specific PCR ??
ThX
Hi lactamase,
u can use NCBI's primer blast programme to find out if ur primers are aligning with any gene other than that of your interest. even if there is misalignment of 5-6 bp at the extending end with a non-specific gene, its fine to go ahead with the primer. Inspite of all this, if you still get non-specific amplifications, then u need to fiddle with the annealing temperature. see if increasing the anealing temp with the same set of primers helps.
all the best!
You can also titrate the Mg concentration, run a gradient PCR (assuming your machine can do one) or try a touchdown PCR.
How did you calculate the Tm? Try your sequences on a few sites (just google for them), and you'll get a few answers! I just use 2(A+T) + 4(G+C); works most times!