7 replies to this topic

[Neotoma]

I have been running a PCR on a nuclear gene from genomic DNA and it has randomly stopped working.  I ran a positive control using gDNA that I know is good, with primers elsewhere that I know are good, and I am getting a product, so I know it is not a problem with my thermal cycler, tubes, tips, template or reagents (except primers).  I have been using the same primers for months and have been getting a product, and I am using the same stock primers as before, the same taq, the same everything but I can't get a product now.  I just get a long smear.  Any tips on troubleshooting???

[phage434]

Same template?  Same template DNA preparation?  Same dilution of template DNA?  Have you tried dilutions of your template?

[gebirgsziege]

up on what was already said....order new primers? or is something wrong with your thermocycler? And have you checked if the PCR-programm was changed; there are people who just change annealing temps or times if they quickly need to test new primers etc.

Edited by gebirgsziege, 17 February 2010 - 03:36 AM.

[Baars01]

Perhaps organic compounds during DNA isolation ended up in your PCR reaction mix. Have you performed a 260/230 test?

[Neotoma]

As I said before, I am using a DNA template that I have gotten to work before under the same conditions, so I'm not sure whether changing the concentration will help, although I can try.  Also, my thermal profile has not changed, that was the first thing I checked.  A260/230 is 1.93, although I'm not sure how to interpret this?

[Stuck with Ligation]

I had this problem for a whole week. Easiest way to solve it:

1. New Sterile Water
2. New dNTP Mix
3. New template
4. New Polymerase (if other PCR's don't work)
5. Clean pipettes in 70% ethanol and then wipe down. Or autoclave

When I went with all new materials my PCR's started working like a charm.

[Neotoma]

Ok I'm running a PCR with all new reagents and I'll see what happens.  I don't think its the pipets because I have been using filter tips which have not helped, and no one else in the lab is having my same problem.  I'll let you know.

[Baars01]

Don't worry, 1.93 is very good. 2 is optimal, <1.4 is poor, although PCR can still work, depending on the manufacturer