What else can I do with my PCR?

[studentX]

Hi, I have new primers (24bp, Tm 60, GC content 50%), blast said that they are specific to my target sequence, but, they dont work and I have no idea why. The problem is, that I need 500bp band,and I still have only strong 200 and 300 bp bands. I´ve tried temperature gradient, touchdown PCR, glycerol, DMSO and BSA. So my question is, if it is possible to do anything else, or only to redesign primers? Thanks.

[JackBean]

what is your teplate? Plasmid, gDNA, cDNA/mRNA? What conditions do you use? What about other polymerase?

[studentX]

I used genomic DNA (from blood and tissue), 50ng. Polymerase was from Fermentas, recombinant Taq polymerase, and MasterMix (Fermentas, 4xMgCl2)...my basic PCR profil was 3 min. denaturation, 30sec. denaturation, gradient 55 - 65C 30sec., 72C 30 sec. and last step 72C 10 min. Then I changed gradient, increased extension time and temperature, changed primer concentration and MgCl2 concentration...

[JackBean]

did you try to increase/decrease the amount of template? Or try to digest it with some RE? Best if you know the sequence, you could find some RE, which does not cut in your region.

However, I don't know, how much amplification you get of size 200 and 300 bp. If is it really a strong amplification, than you should probably sequence it to see, whether is it something completely different or related to your amplicon...

[studentX]

OK, thank you for your answer, I will try to do something with it again and I will see. (I cannot sequence it, I am a student, I dont know where and how to sequence it). Maybe one day....

[clevermizo]

I would strongly suggest varying the amount of starting template. I've used above 100 ng genomic DNA to seed PCR. 50 ng cuts it for plasmids, but with genomic DNA remember that it's not 50 ng of your target sequence, it's 50 ng of the entire genome. You need to increase your odds a little. I would just do a very basic PCR program at one annealing temp, maybe one adjuvant like <10% DMSO, and vary template: 50, 100, 200, 300 ng and see if this helps.

[JackBean]

of course, that can help, but too much templeta can be inhibitory, as the sequence will be lost...

[clevermizo]

Well it sounds like template amount is the only thing that the OP hasn't tried to optimize yet, so it's probably worth a shot.

[JackBean]

I don't say no I have recommended it as the first thing