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PCR: Band in one lane, streaking in the other..HELP!

Started by Doggie Science, Jul 08 2011 07:47 AM

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3 replies to this topic

#1 Doggie Science

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Posted 08 July 2011 - 07:47 AM

Hello all,

We are doing a PCR (1.2KB amplicon)with genomic mouse DNA and a vector to simulate a positive genotyping PCR. Vector is diluted so there is one copy per genome. 50ng of genomic mouse DNA are used.

Our problem is that we get streaking in some lanes, and the specific band in others. Picture attached.

What we have tried:

-New reagents
-New genomic DNA
-New diluted vector
-Fresh TAE to run the gel
-Fresh SYBR gold to stain the gel
-Using a different thermocycler

In the picture, everything is from the same master mix... so we really don't know what to think.

Any help is appreciated!

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#2 Adrian K

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Posted 08 July 2011 - 09:33 AM

Try do a gradient dilution of your genomic DNA. I think your genomic DNA is way far too much...

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#3 Ameya P

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Posted 11 July 2011 - 04:26 AM

Second what Adrian has said.

Too much DNA.

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#4 Doggie Science

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Posted 12 July 2011 - 07:00 AM

Adrian K, on 08 July 2011 - 09:33 AM, said:

Try do a gradient dilution of your genomic DNA. I think your genomic DNA is way far too much...

gt_ameya, on 11 July 2011 - 04:26 AM, said:

Second what Adrian has said.

Too much DNA.

Thanks for the response guys.

We have tried a control with just the vector (no genomic),and we still get the hit or miss streaking. We will try less genomic also, but we are know thinking it could be hyper coiling of the vector. We are liniarizing that.

What do you think? Any other ideas?

Thanks a bunch!