PCR band for water but no contamination, Digestion not working - (Sep/28/2006 )

Hi,
I have the following problems with this project that I have been working on for two months now and it's not going anywhere.

1. I get a band for water.
I know the water band is not due to contamination. I wanted to check if my primer is contaminated so I used my forward primer as a template (and another set of primers as primers) and I got a band. I got a new sample of the same forward primer from the company and again the same thing happened.
But there is no band when I use the reverse primer as a template. In fact, I get a band for water and the forward primer but absolutely no band for the reverse primer.

2. Genomic DNA digestion doesn't work
I am digesting my PCR product with Hinf1 and I am supposed to see a digested product which has a band difference of about 20bp from the undigested product. I run this on a 2% TAE-agarose gel (70V, 1 hr) and sometimes I am able to see this, but it is not reproducible. The same sample gives me different results each time I repeat it, even though I do the exact same thing everytime.

Please share your thoughts on what you think could be going wrong here. Thanks very much.

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what size is your water band?

I am thinking it's primer-dimer

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I don't know what to say. This experiment detail sounds odd.

I would have just added either the forward primer or reverse primer to the PCR reaction mix to determine if either primer was contaminated with template. I don't see what a second set of primers would do, especially if they use the either the forward primer(pF) or reverse primers(pR) as templates.... (and that would mean pF and pR are rather large primers to even work as templates.)

But in any case it certainly would appear that that the water control band, is caused by some kind of self annealing with the forward primer (primer dimers? perhaps). If the water control band is unacceptable, I would suggest either a redesign of the forward primer, or a change in annealing conditions... rasing the temperature might help.



? <Lifts hat and scratches head>
Must be a spelling mistake.... in any case, it looks like somekind of instability with the digestion mix.

But first... regardless of the digest, does the PCR reaction give approximately the same amount of DNA for each run/sample? Do you notice any lost of volume (ie concentration of salts) of the PCR reaction mix after the thermal cycling is completed?

Could you write down the digestion formulation that you use to cut your PCR DNA.

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1. I thought it was a primer-dimer too. But the water band is about 235bp. The primer-dimer should be much smaller, right?
No, the primers are not large enough to act as templates - they are about 22bp.
I am going to try increasing the annealing temperature, like you suggested. Thanks!

2. I digest the PCR product taking 1ul Hinf1, 1ul buffer and 8ul DNA and incubating overnight at 37 degrees.

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One more thing, yesterday I did the digestion again and on the gel, the digested sample should have two bands at 215 and 20bp. The undigested sample should have one band at 235bp. But for the digested sample, there was just one band at about 235bp (same as undigested), and it was fainter than the undigested band. This has happened several times before. Is the digested faint band anyway implying that my sample has got digested but my gel % is not enough for a 20bp resolution?

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did you check if the other components of the reaction are clean?

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ye, may be the gel resolution is not enough..
but can't be your faint band represent a small percentage of your sample uncut, i mean incomplete digestion!?

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Yes the other components are clean, since they have worked in other PCR's.

Could the faint band be incomplete digestion as Strawberry suggests? Will it be overcome by increasing the amount of restriction enzyme?

Thanks.

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Maybe. Yes, do increase the amount of restriction enzyme. But I would also increase the volume of the digest. A 10ul digest overnight, could be very prone to evaporation problems. Which would change the salt concentration of the digest.

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Have you consider that one of your reagents may be contaminated with a really tiny amount of DNA (1ng)?

If that's the case, it may be that only those primers amplify that sequence enough to be seen in a gel and the other primers don't bind to that sequence in the rest of the PCR.

About cheking your primers that way, wouldn't it be easier just run 20ul of your stock solution in a gel and see if there is a extra band? Don't you use working concentrations of your primers? What I do if I get contamination is first get rid of all the working concentrations used, make new ones and perform the same PCR. It usually works most of the times.

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