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gel purification/ta cloning issues

gel purification/ta cloning issues - Molecular Biology Techniques

gel purification/ta cloning issues - Molecular Biology Forum. Includes forums for common molecular biology techniques.

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Old 05-19-2010, 08:25 PM
Pipette Filler
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Default gel purification/ta cloning issues

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.

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 purifiying 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?

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