2 replies to this topic

[lactamase]

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

[Vini]

lactamase, on May 20 2009, 03:44 AM, said:

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

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!

[swanny]

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!