Weird PCR inconsistency

Hi,

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i agree with it too.

I agree with aqua plasmid. Most often researchers face such problems and come to forums to solve them. This is the reason Journal of Errology link is created, so as to document all problems faced during the process of a research and the solutions adopted.

I would like to request all researchers to please upload their problems and their solutions on www.bioflukes.com, even if these problems are already available in forums.

Along with these problems, we also accepts futile hypothesis, errors faced and other valuable data from empherical research.

I would like to request moderators to also submit iconic problems which they have solved in the past.

I know this is a old thread. Bszekely may have already solved his problem. But it may help others to know that fecal DNA is often contaminated PCR inhibitors. If using diluted DNA (down to ~300 genome copies for 30 PCR cycles) would not help, then fecal DNA kits that remove PCR inhibitors will be needed for consistent PCR results. I'd like to suggest our AquaPrecipi link reagent, which remove PCR inhibitors from fecal materials.

Yes, you are correct. I can't get 4 reactions to work after I've done the test where 25 ng works and the 50 and 150 doesn't. I haven't changed any reagents and I have noted which wells I am using, but that is a possibility that the wells aren't consistent. The band is in the right place, which is about 1500 bp. I did another test today and will run a gel tomorrow. Hopefully, I'll get some more results soon. Thanks for the response. Has anything like this every happened to you?

I have definitely seen strange things happen from PCR machine to PCR machine, but not typically in the same machine. This has happened where I set up the reactions in duplicate and get a nice band in one machine but not the other.

Is there any chance the machine you are using is doing a gradient for some reason? So the block is not uniform.

Also, if your band is 1500bp, you probably do not need to be running a 1.5% gel. I would go with 0.8%-1% gel for a band that size.

It sounds like the new prep may have some contaminant that is inhibiting the PCR reaction. You might want to try cleaning up the DNA with a phenol/chloroform extraction or a clean-up kit of some sort. It would also be good to sequence the band you get just to make sure it's what you think it is. Finally, is there some housekeeping gene you could amplify to check things out? For RT-PCR of mammalian mRNA, I routinely run a PCR with GAPDH primers to see if the cDNA reaction worked. If I get no band, I reprecipitate the RNA and try again. If still no band, I toss it and start over.

Have fun.

mike

Yes, you are correct. I can't get 4 reactions to work after I've done the test where 25 ng works and the 50 and 150 doesn't. I haven't changed any reagents and I have noted which wells I am using, but that is a possibility that the wells aren't consistent. The band is in the right place, which is about 1500 bp. I did another test today and will run a gel tomorrow. Hopefully, I'll get some more results soon. Thanks for the response. Has anything like this every happened to you?

First, for my new sample, I did 100 ng of DNA/25 ul reaction (as it always worked before) and no bands showed up. I then did a test and used 25 ng, 50 ng, and 150 ng each in a total volume of 25 ul. Only the 25 ng tube showed a single band (which was in the correct place also) on my 1.5% agarose gel, while the 50ng and 150ng showed no bands. I thought this was great so I did 4 reactions each with 25 ng / 25 ul reaction. NO BANDS showed up on this run. I did the 25 ng, 50 ng, 150 ng test again, and the same thing happened as before.......one aweseome single band for the 25 ng tube, and no bands in any of the other samples. I then tried a sample that worked previously and that worked fine, so my reagents are most likely just great, but I can't get multiple reactions to work. Any thoughts?

Wow, that sounds incredibly confusing.

So when you do the concentration test you get a band with 25ng of template, but not with 50 or 150ng.

However, when you set it up again using 25ng but with 4 separate tubes, none of them work. Is that correct?

Are you sure the band is the correct one you are looking for? I know you mentioned you are running a 1.5% gel, are you trying to amplify a very small product?

Did you use the same thermal cycler and conditions all three times? Did you use a different polymerase? Did you use the same wells in the thermal cycler? Maybe your block is not heating evenly and some wells are working better than others. If your PCR is not very efficient, this might cause problems.

It sounds like something strange must be going on.

I am trying to perform PCR on extracted bacterial DNA from feces. I have done PCR on several samples and can get previous samples to work. Typically, I've been using 100 ng of DNA in a total volum of 25 ul for a PCR of 30 cycles and everything has been just great.

First, for my new sample, I did 100 ng of DNA/25 ul reaction (as it always worked before) and no bands showed up. I then did a test and used 25 ng, 50 ng, and 150 ng each in a total volume of 25 ul. Only the 25 ng tube showed a single band (which was in the correct place also) on my 1.5% agarose gel, while the 50ng and 150ng showed no bands. I thought this was great so I did 4 reactions each with 25 ng / 25 ul reaction. NO BANDS showed up on this run. I did the 25 ng, 50 ng, 150 ng test again, and the same thing happened as before.......one aweseome single band for the 25 ng tube, and no bands in any of the other samples. I then tried a sample that worked previously and that worked fine, so my reagents are most likely just great, but I can't get multiple reactions to work. Any thoughts?