PCR with DMSO - (Dec/01/2004 )
QUOTE (robyn @ Jun 13 2007, 09:46 AM)
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
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
Could you tell me what different concentrations of MgCl2 you tried? I'm doing the same thing, but I'm not sure how much I can vary the concentration. Someone help?
-Dubs-
QUOTE (perneseblue @ Jun 28 2007, 09:32 PM)
QUOTE (vetticus3 @ Dec 6 2004, 07:05 AM)
what doesn't kill me can only make me stronger.
Except a horrible disfiguring accident that leaves you paralysed for life
Though, best wishes on having defeated the monster of the week! So what made the reaction work again? Was it the fresh DMSO made from the pure DMSO solution?
It can be correct that the DMSO inhibit the taq polymerase during the PCR. But I am telling you, I have done PCR many many times using DMSO and Taq at a regular concentration and the reactoin went beautifully. Why would DMSO inhibit the enzyme one time and not the other? Confuisng!
-mabusheh-
most likely due to the purity of said DMSO.
I have learned that 99.9% pure can mean absolute purity - 99.9g DMSO and 0.1g rubbish. Or it can also mean once vapourised, said DMSO vapour is 99.9% pure. Which mean the absolute purity can be far lower.
And finally the 0.1% impurity for two different companies may be composed of different contaminants.
-perneseblue-