Aaaah I want to die!!!! PCR won't work - Why do the extractions that amplified 2 weeks ago fail now? (Apr/12/2010 )

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I would first test new dNTPs and primers, these are the PCR components that degrade most commonly, before suspecting the samples though.

Research Papers

-William Parkar-

William Parkar on Apr 21 2010, 10:23 PM said:

I would first test new dNTPs and primers, these are the PCR components that degrade most commonly, before suspecting the samples though.

Research Papers

It is VERY, VERY unlikely that the sample is degraded. Even if it was heavily degraded (which would require some effort on your part) you would probably be able to get a PCR to work on it, depending on the size of course. In all my years in the lab, I have NEVER had a problem with DNA degrading. You could leave it on the bench for weeks and it will still work. The sample always becomes the scapegoat because it is easier to blame that than something else.

More than likely something else has happened and it is more than likely related to something you have done/changed.

-Dukey-

vladooo on Apr 14 2010, 04:44 AM said:

AND: DNA does NOT degrade when frozen (almost). Look at as they found mammoth DNA 37 000 years old. Your life is too short to detect any degradation of frozen sample. <_<

Hi All,

For those who may read this post later on down the line (when your own PCR is not working, and you want to die) -- vortexing the thawed samples thoroughly was extremely helpful. I thought I was going to have to reextract everything -- but it turns out you just gotta vortex the crap outta those tubes. Changed everything! Thanks forum members!!

Kim

-KimWG-

KimWG on Apr 30 2010, 07:30 PM said:

vladooo on Apr 14 2010, 04:44 AM said:

AND: DNA does NOT degrade when frozen (almost). Look at as they found mammoth DNA 37 000 years old. Your life is too short to detect any degradation of frozen sample.

that's great. I won

-DocFlow-

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