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PCR has stopped working


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7 replies to this topic

#1 Neotoma

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Posted 16 February 2010 - 02:05 PM

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???

#2 phage434

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Posted 16 February 2010 - 04:58 PM

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

#3 gebirgsziege

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Posted 17 February 2010 - 03:34 AM

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.

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#4 Baars01

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Posted 17 February 2010 - 03:50 AM

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

#5 Neotoma

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Posted 17 February 2010 - 07:23 AM

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?

#6 Stuck with Ligation

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Posted 17 February 2010 - 10:44 AM

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.

#7 Neotoma

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Posted 17 February 2010 - 12:22 PM

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.

#8 Baars01

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Posted 18 February 2010 - 07:59 AM

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




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