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PCR DIG-labeling kit preblem?


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#1 CandyTon

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Posted 13 May 2015 - 01:26 AM

I've been having a frustrating time trying to produce a DIG-labelled DNA probe using the Roche PCR DIG labelling kit.

With ordinary dNTPs, the PCR works well with both my template/primer combination and the positive control template/primer combination supplied with the kit. I get the expected sized PCR products on a gel stained with gel red and visualised under UV.

However, when I use the DIG synthesis mix, which contains dNTPs plus DIG-dUTP, I get nothing that is visible on a gel. The result is the same with both the Roche positive control template and my own template. When I measure both PCR products (the one produced using dNTPS and the one produced using the DIG mix) on a nanodrop, they both measure the same concentration (about 410 ng/ul).

It seems that the PCR with the DIG-labelling mix may have worked ok, but there is nothing at all visible on a gel. I suspect that the product is there, but it isn't staining with gel red. The Roche protocol recommends staining with EtBR, which we no longer have in our lab.

Has anyone come across this before?



#2 bob1

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Posted 13 May 2015 - 01:45 AM

It won't be the gel that is the problem. The DIG labelled PCR products have different reaction kinetics to ordinary PCR, you could try a lower temp annealing, especially if you are generating the probe off something specific like a plasmid.

 

Checking the products on a nanodrop doesn't tell you anything; dNTPs should give you a reading all by themselves.






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