4 replies to this topic

[buccal_dave]

Hello Everyone,

Out of the blue I started seeing a smear on my gel from my pcr product. I have been using the same primers, the same reagents, and the same settings on my thermocycler. I saw the same smearing a month ago, and decided, after weeks of frustration, to buy all new reagents (the same brand of everything, since my pcr has worked perfectly in the past).

Background:

I am using bisulfite converted DNA that is extracted from buccal swabs. I then do a nested PCR with the first amplicon being around 340bp and second being around 200bp.

my pcr setting for both pcrs:

95c 15m
95c 30s
54c 1m30s
72c 2m
40 cycles
72c 10m

I am seeing the smear after I run the first PCR (340bp) on the gel. I have processed and sequenced hundreds of samples using this technique, but it suddenly stopped working and I am confused. I attached a picture of the gel Anyone have any ideas????

Attached thumbnail(s)

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[Michaelro]

Hi
It looks like some nuclease contamination or primer degradation.
Nuclease contamination can originate from your primer stock or your template DNA (since all other reagents are newly ordered)
If you're sure your template DNA is OK - I'd order fresh primer stock.
Good luck
Michael

[ElHo]

Your converted dna could be degraded. If possible check with another pcr for converted dna established in your lab and use positive controls (commercial or cloned converted DNA) to further localize the problem (pcr or DNA).

[buccal_dave]

Michaelro, on 05 August 2010 - 12:13 AM, said:

Hi
It looks like some nuclease contamination or primer degradation.
Nuclease contamination can originate from your primer stock or your template DNA (since all other reagents are newly ordered)
If you're sure your template DNA is OK - I'd order fresh primer stock.
Good luck
Michael

I have replaced my primers with a new company, but the same sequence, and am still seeing a smear! I know that my DNA is okay because I am able to amplify a smaller band within the larger region (second pcr of the nested). I have replaced everything at this point, and I don't think it is contamination because I don't see anything like with the second set of primers. I dont know...

[Biog]

buccal_dave, on 17 August 2010 - 07:26 AM, said:

Michaelro, on 05 August 2010 - 12:13 AM, said:

Hi
It looks like some nuclease contamination or primer degradation.
Nuclease contamination can originate from your primer stock or your template DNA (since all other reagents are newly ordered)
If you're sure your template DNA is OK - I'd order fresh primer stock.
Good luck
Michael

I have replaced my primers with a new company, but the same sequence, and am still seeing a smear! I know that my DNA is okay because I am able to amplify a smaller band within the larger region (second pcr of the nested). I have replaced everything at this point, and I don't think it is contamination because I don't see anything like with the second set of primers. I dont know...

And what about program settings ? I mean UV and image-gel capture software in your lab? Are you sure there is no modification in its setting before you pass on?