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My PCR suddenly stopped working and I'm losing my mind

pcr

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#1 Okazaki_Fragment

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Posted 11 December 2014 - 02:09 PM

I've been (unsuccessfully) trying to run PCR for BMAL1 and Adip-Cre for about a month now. I started running both genes in August without any problems, but somewhere around the middle of November something horrible happened and I've either been getting no bands, faint bands, or false positives. I've replaced all of my reagents, I've re-isolated my DNA, I've used different thermo cyclers, what do I do next?? Another tech in my lab is having the same problem. Please help, I'm about to sell my soul for a working PCR.

 

Here's my info for BMAL1:

10* PCR Buffer: 2uL

25mM MgCl2 1.2uL

dNTP's (10mM) 0.4uL

6013 (10uM) 0.4uL

6014(10um) 0.4uL

5436 (10uM) 0.4uL

Taq Polymerase 0.2uL

5mM Cred 2.5uL

dH2O 10.5uL

Total 18uL

 

And for Cre:

10* PCR Buffer 2uL

25mM MgCl2 1.2uL

dNTP's (10mM) 0.4uL

GAF008 (10uM) 0.4uL

GAF009 (10 uM) 0.4uL

Taq Polymerase 0.2uL

5mM Cred 2.5uL

dH2O 10.9uL

Total 18uL


Edited by Okazaki_Fragment, 11 December 2014 - 02:11 PM.


#2 bob1

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Posted 12 December 2014 - 01:02 AM

I'm not sure what the 5 mM Cred is, but that aside. It looks like you are using 2 ul of DNA, but you don't say how much this is in total. Too much DNA can cause PCRs to fail, as can contaminants in the DNA extraction. Try diluting your DNA 1:100, 1:500 and 1:1000 and see if it works.



#3 Okazaki_Fragment

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Posted 12 December 2014 - 07:19 AM

Thanks for the response! Cred is is Cresol red. That's correct, I'm using 2 uL DNA. It's usually around 70-80ng, but sometimes can get up to 120ng. I'm still new to PCR, could you tell me how much DNA is "too much"?


Edited by Okazaki_Fragment, 12 December 2014 - 07:20 AM.


#4 bob1

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Posted 12 December 2014 - 10:23 AM

It depends on the organism. A general guide is: a maximum 500 ng of human genomic DNA, 1 – 10 ng of bacterial DNA, 0.1 – 500 ng of plasmid DNA. However, the contaminant problem is often seen when using undiluted DNA, so have a go with the diluting.
 







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