PCR with DMSO - (Dec/01/2004 )

Hi,
I've said this before, but, basically, i've been using a PCR reaction with Pfu on genomic DNA, and until very recently, it's worked really well. last few weeks, the reaction flopped majorly. Other researchers in our lab have used the Pfu also, unsuccessfully. So, we got a new batch out of our freezer, it too didn't work. Changed the water... didn't work. Fresh dNTP... didn't work, fresh primers... didn't work. changed the PCR machine from my floor to the one upstairs... didn't work. swore... didn't work. changed from pfu to another proofreader with it's very own buffer and with assurances that this PCR would work... didn't work (though did get some lovely primer dimers)....
I was wondering... and was hoping someone could confirm this.... the only thing that has remained constant is my supply of DMSO (5%). could this be the thorn that screwing me around? (as i write this, i'm running a PCr with a fresh batch of DMSO)

can DMSO go bad?

hoping desperately that this reaction works.

supposing this reaction doesn't work, and i manage not to throw myself off a building, is there anything else i can try. this reaction has worked for months, and now is not.

vetticus

fresh DMSO... didn't work. i don't get it. I got this reaction to work, One day it's fine... next it's stuffed. Which molecular biology god should i make a sacrifice to?

Hi

I am fairly sure that DMSO degrades slowly in the presence of water, it could be that the 5% solution you are using is degraded and seeing your last comment, it could be that the new stock is also degraded if stored as 5%.

Just a thought- try fresh DNA, if possible, it is barely likely but it could be the factor as it seems to be the only one not changed.

Otherwise I am flummoxed!
Good luck
Bob

apparently DMSO degrades if exposed to sunlight... ahhhh. still doesn't help me out (but usefull for anyone else out there). dont' store it at 5%, that's the percentage of final volume... my head hurts.
trying fresh DNA... will probably have result in ~30 min.
thinking about it all day... the only other variable that has changed is that the other lab swiped our radio and is playing pop rock instead of our usual indy/alternative. i don't know. perhaps i should stand on one leg whist preparing the reaction.

there must have been something wrong with your DNA template. please store the template at 4 centigrade but not freeze them. good luck!

It works... it works.... it works... little dance little dance.... it works...
what happened... i offered a blood sacrifice to one of the more fickle gods of molecular biology (ok, maybe not, but the idea did cross my mind)... seriously, i have no idea what happened. it just stopped working, but it works now...little dance little dance. wearing my lucky red t-shirt must have pushed it over the line.
the dna's fine, the primers are fine, the mg content is fine, the water is fine, the machine is fine.... little dance little dance little dance.
what doesn't kill me can only make me stronger. can finally breath normally again.

It works... it works.... it works... little dance little dance.... it works...
what happened... i offered a blood sacrifice to one of the more fickle gods of molecular biology (ok, maybe not, but the idea did cross my mind)...  seriously, i have no idea what happened.  it just stopped working, but it works now...little dance little dance.  wearing my lucky red t-shirt must have pushed it over the line. 
the dna's fine, the primers are fine, the mg content is fine, the water is fine, the machine is fine.... little dance little dance little dance.
what doesn't kill me can only make me stronger.  can finally breath normally again.

Hi guys

I am also having the same problem! I am trying to amplify exon 4 of the caprine kappa-casein gene with primers I got from another article. 2 months ago I tested the PCR reaction with the temperatures they gave (Ta = 63). It worked ok. A colleague suggested that it would work better at Ta=67. I tried that - it worked beautifully!!!

Now, I want to integrate the PCr step into the rest of the steps and suddenly at Ta=67 there is nothing but a few weak bands and hectically bright primer-dimers! I know what you're thinking: old ingredients, or it must be some mistake. Not so. I tested it again carefully at 67 - no luck. Tested it with new primers and new dNTPs and new water - no luck. Decided to play around with the temperature - tried 65 (so-so), 68 (nothing), 62 (so-so, less specific bands and still have primer-dimer). Now I dont know what to do. I dont know if I should start messing around with MgCl2 concentration because it DID work. Someone mentioned that my DNA may have degraded from being stored in the fridge? Any help will be greatly appreciated as I dont have any more hair left to pull out!!

Robyn