gel purificatino and/or ta cloning issue

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foster033
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gel purificatino and/or ta cloning issue

Post by foster033 » Fri May 21, 2010 8:01 pm

Hi Everyone,

I'm trying to determine a novel mutation on pt. dna. I'm using gene specific primers to amplify what should be a 637bp band in normal dna. Using these primers I get 4 bands from the pt. dna (as well as the carrier mother). They are 468 bp, 637bp,~900bp, ~1400bp. The 468bp band is a confirmed 169bp deletion but I'm unable to determine what is causing the two larger bands. They do not appear in positive or negative control reactions. Since there should only be two alleles I'm thinking these larger bands are a result of a complicated duplication/deletion mutation but I've been unable to isolate the larger two bands to determine where they are coming from.

After TA cloning this pool of products only the 468bp and 637 bp band show up after colony pcr w/M13F/R primers. My PI suggested doing a miniprep instead of colony pcr but I don't see how that could help, the problem is with the larger bands getting into the vector not the colony pcr, as there really aren't any negatives in screening.

I've tried gel purifying all 4 bands and then TA cloning from the gel purified products but even when I only use the gel purified dna from the larger two bands the TA clones only show the smaller bands after colony pcr (>95% deletion product).

I'm positive that the four bands are isolated separately but when rerunning them on a gel or cloning I always get only the smaller products and I'm unable to isolate the two larger bands in any way.

It seems like the are some type of artifact (from the deletion?), but they are present in both pt. and pt's mother dna and not present in wt dna or neg control.

Has anyone ever had a similar problem or have any ideas how I can determine what these two large bands are?

Thanks!!

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canalon
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Post by canalon » Fri May 28, 2010 1:04 pm

Never seen anything like that.
It seems that the smaller product are contaminating those bands too. You can try to increase the relative concentration by PCR them again from the first gel : just pick in the band with a 10µl tip and mix it in a PCR mix with all reagents, that should be enough DNA to be used as template. See what happens if you PCR again: Pure large bands or again a mix. You can repeat again and again (I have seen paper where they do it up to 6 times to separate different products that are moving very close to one another, but I would be careful because of the risk of accumulation of errors by the PCR).
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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