Hi everyone
We've been trying to sequence our region of interest from some patients. The genomic DNA they've sent us is not of great quality, but PCR works so they musn't be terrible.
I amplify the region of interest from genomic DNA, I check on the agarose gel... there's my expected DNA band. Great. If we sequence this directly, it's too dirty to get good results, as expected.
However, when I purify the samples with spin columns (I've tried two different brands) sequencing is not possible, they just get no signal, and samples are indeed quite dirty post-purification... but we've been using these columns forever, we've purified and sequenced tons of samples... any idea other than ask for better-quality genomic DNA?? I've already checked on an agarose gel post-purification so yes, DNA is still there.
We've tried:
REF 740609,50 Machery-Nagel, Nucleospin Gel and PCR clean-up
ISOLATE II PCR & Gel Kit, Cat No. BIO-52059
Thanks in advance
Bea
Judging by the absorbance data, there is a lot of organic reagent and/or salt contamination (most likely guanidium salts) in the sample that may be interfering with your sequencing PCR.
Washing the spin column twice, after DNA binding, with the ethanol-containing wash buffer should minimize salt contaminants
Alternatively, you can clean up your samples or the genomic DNA by performing a phenol : chloroform : isoamyl alcohol separation, followed by ethanol precipitation. The phenol/chloroform separation can remove most of the organic contaminants. Take care while pipetting to make sure there is no phenol/chloroform carry-over. During ethanol precipitation wash the pellet thrice with 70% ethanol to remove the excess salt.
You could skip the phenol/chloroform extraction if you know that its only salt contamination. Just ethanol re-preciptation and washes with 70% ethanol would remove the salt.
Did you send raw DNA for the gene sequencing? If you yes, I will suggest you also send PCR product for the sequence and check your primers of your target genes.
Also check the concentration of the DNA.
Thank you.
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