PCR Setup stopped working - (Oct/08/2006 )
I have been using a PCR setup that had been working well for about a month, and it suddenly stopped working about two weeks ago (i.e. we had a clear, clean band of the expected size, and now we have no bands seen in the resulting gel).
Here is what I've done so far to try to fix this:
1) all reagents have been replaced - new Taq, dNTP's, buffer, and primers (same brands and
primer sets used previously, just fresh)
2) all materials used have been freshly autoclaved - tips, dH20, and tubes
3) I've borrowed someone else's thermocycler to run the reaction
4) I've tried different DNA extractions (I've used brand new extractions as well as one's that I had
stored that had worked previously). New extractions were with a brand new kit.
5) I've setup both fungal and bacterial PCR's that have previously worked, but no luck. I've
now used 6 different previously working primer sets.
6) I had a different lab member setup reactions to make sure it wasn't something I had started
doing incorrectly.
At this point, I'm looking for any new ideas for what might be causing my problem. Any help at all would be appreciated - no idea is too simple. Thanks in advance!
Here is my basic setup for my master mix:
Taq 0.25ul TAKARA (from 250U)
Buffer 2.50ul TAKARA
dNTP's 2.00ul TAKARA
Primers 1.00ul ordered from IDT (10pmole/ul)
dH20 16.25ul
DNA 2.00ul I have no way to measure concentration of this, but as I said, it had been
working previously (from MoBio Soil Extraction kit)
I need two more bit of information,
1 - Have you confirmed that there is good quality DNA in the extraction. (by running the raw DNA on a gel)
2- Is this PCR problem limited to only this reaction? Meaning, are any other PCR reactions by yourself or other labmates working?
Other things to check: water quality (often overlooked), gel loading dye contamination, gel running buffer contamination. Can you run gels and see bands? Does your marker lane show up? If you mix your marker with your loading dye does it still show up? Are the caps on the tubes the same? Have you set the cycler to use the hot lid (one of my students had problems for a week with nothing working because the hot lid was turned off).
1 - Have you confirmed that there is good quality DNA in the extraction. (by running the raw DNA on a gel)
2- Is this PCR problem limited to only this reaction? Meaning, are any other PCR reactions by yourself or other labmates working?
1 - I have confirmed, and I can see genomic DNA although it is weaker than expected.
2 - All PCR's we had been working on (a set of 6) have stopped working; there are two of us
setting up these reactions and nothing at all has worked for two weeks now.
Freshly autoclaved water taken from a millipore system has been used as well as eppendorf water straight from the box. Our marker can be seen on all the gels, and we are using the same loading dye with it as we are using to load the samples. We have used both TAE and TBE running buffer freshly made. Same caps. Hot lid turned on.
Thanks for all the ideas everyone.
You might be having inhibition from soil components, or from excessive amounts of DNA. I would try serial dilutions of your DNA 10x, 100x, 1000x.
At this point, I would be trying to find someone else in a neighboring lab who had working PCRs and trying hard to replicate the exact ones in my lab with my equipment and reagents. Substitute theirs for yours one at a time until you locate the problem. I'm guessing that one or your reagents is still bad.
Have you tried with lower template DNA amount in the reaction mixture and higher number of PCR cycles. If the extracted DNA was ungood enough (some DNA extraction can't eliminate all PCR inhibitors such as SDS or others), lowering the DNA amount will also reducing the amount of inhibitors, so increaing the PCR efficiency. When we use DNA extracted from soil, PCR often fails with DNA from original extracts, while PCR almost always succeed with 100 fold diluted DNA from original extracts.
sounds like you have a system wide failure.
phage434 and zhongmindai have made some very good suggestion
-of diluting DNA with water, to sidestep DNA inhibiting agents in the template
-running totally on another labs PCR reagents and equipment. See if that help.
- and looking at the water.
I only want to expend on the water point. Have you checked the filtration cartridges on the millipore system? Do those things need replacing yet? Is the membrane still okay. Was there any recent work done on the piping within the building.
At this point, I would be trying to find someone else in a neighboring lab who had working PCRs and trying hard to replicate the exact ones in my lab with my equipment and reagents. Substitute theirs for yours one at a time until you locate the problem. I'm guessing that one or your reagents is still bad.
Although we use MoBio's Soil Extraction kit, we are actually just pulling the extract from a colony on a plate. Regardless, I did a dilution series on an extraction that worked two weeks ago this morning. I still have no bands showing up.
We have replaced all the reagents to our knowledge and are now moving on to different brands at this point. I think finding another lab to borrow for a day might be our best idea at this point as well. Thanks for the input.
Thanks for the suggestion - I ran a serial dilution this morning with the DNA diluted up to 1000 fold and still no luck.