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Looking for help with my PCR!


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#1 milla

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Posted 23 November 2009 - 02:46 PM

Hi everybody!

I need some help with my PCR.

I am getting a kind of smear... but really strange (I have attached a picture!).

Do you think I have to try make some change like when you have a normal smear or the problem could be something else?

Thank very much!

Attached Thumbnails

  • pcr.jpg


#2 ivanbio

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Posted 23 November 2009 - 03:03 PM

Hi milla,

The image you posted suggests to me that most of your DNA remains stuck in the wells. Since your size marker runs just fine, my guess is that there is something in your template that is inhibiting migration; protein contamination perhaps? If you are using a lot of template DNA in your PCR reaction, then this could be the case.

The smear you see, especially in wells 2 and 4, suggests degradation.

Either way, I would suggest diluting, or cleaning, your template DNA and see what happens. Running your DNA by itself on the gel would also be a good idea. If you do not see a nice tight band towards the top of the gel, then your genomic DNA template may not be of good quality.

Good luck

Ivan
Carlsbad, CA

#3 lotus

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Posted 23 November 2009 - 03:05 PM

Repeat the PCR with a positive control that you know definitely works. Then run the gel again. It looks like there is too much DNA in each lane and a lot of sheared products.

Hi everybody!

I need some help with my PCR.

I am getting a kind of smear... but really strange (I have attached a picture!).

Do you think I have to try make some change like when you have a normal smear or the problem could be something else?

Thank very much!



#4 milla

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Posted 23 November 2009 - 03:12 PM

Thanks very much for your suggestions,
I will try immediately!

The DNA is methylated for the bisulfite sequencing... may be this is an explanation of the bad quality of the DNA...




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