2 replies to this topic

[Stuck with Ligation]

I have a PCR reaction that I have run routinely and normally when I clean it up into 50 uL of water I get a conc. of about 25-40 ng/uL. In the last two weeks I have been getting only 10-15 ng/uL which is too little to cut by restriction digest and clean up again for ligation. I have make a new primer working stock from my concentrated store bought primers (stored in -20 for 4 months) and the product has not been remedied. I have also noticed a drastic increase in primer dimers (from none to a bright fuzzy band). Today, I ordered new primers (same as the previous ones that worked great) and am going to repurify the template.

Does anyone have some advice on what is going wrong? This PCR worked great 3 weeks ago and is now not working. I have already switched to a brand new tube of dNTP's and PCR buffer.

[stylothecancer]

Hi,

Have you check your pcr machine? and your pipette?

just wondering...

[Stuck with Ligation]

I checked the PCR machine with another control reaction yesterday (different template) and it worked flawlessly and the negative control yielded no bands...I might order longer primers with a 68-70 degree annealing and try a 2 step protocol.