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No mutation with mutagenesis kit - (Nov/11/2008 )

Hello, I'm new in this field and this site. I really need help on PCR. I've been using mutagenesis kit (Finnzyme and Stratagene) for PCR to get point mutation but no mutation is happening at all. I follow their manual. I get a very faint band on the agarose gel at desired location but DNA sequence comes back as template DNA sequence only. Also I got very thick band way below 0.5kb.

I've used WT DNA template and template which already contain a few mutation. I've tried different annealing temperature and time and different concentration of primers and template. Nothing happening!!! I used 1 ul of each 25pmol/ul primers and 10pg, 100pg, 1ng, and 50ng of DNA template for final volume of 50uL.

I've read about additives but since most of the ingredients are already in the provided master mix, how can I chose the additives (e.g.DMSO) and what concentration should I add??? Is the thick band on the agarose gel primer dimer? What is the possible cause not getting mutation? I'm sure there are my things affecting PCR but would you please give me some advice...any advice. Please!!!

-PrimerX-

QUOTE (PrimerX @ Nov 11 2008, 10:06 PM)
Hello, I'm new in this field and this site. I really need help on PCR. I've been using mutagenesis kit (Finnzyme and Stratagene) for PCR to get point mutation but no mutation is happening at all. I follow their manual. I get a very faint band on the agarose gel at desired location but DNA sequence comes back as template DNA sequence only. Also I got very thick band way below 0.5kb.

I've used WT DNA template and template which already contain a few mutation. I've tried different annealing temperature and time and different concentration of primers and template. Nothing happening!!! I used 1 ul of each 25pmol/ul primers and 10pg, 100pg, 1ng, and 50ng of DNA template for final volume of 50uL.

I've read about additives but since most of the ingredients are already in the provided master mix, how can I chose the additives (e.g.DMSO) and what concentration should I add??? Is the thick band on the agarose gel primer dimer? What is the possible cause not getting mutation? I'm sure there are my things affecting PCR but would you please give me some advice...any advice. Please!!!



Are u performing any control for this experiment,most of the time kit provide a control sample to check....

-bhappy-