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PCR failing


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18 replies to this topic

#16 ocean

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Posted 12 October 2004 - 08:57 PM

hello everyone,

the problem still exists in my PCR...after using a new vial of Taq i still get failing runs? the controls amplify up, but the samples are still failing to amplify..i have tried different buffers and it's not working? can anyone help?

#17 vetticus3

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Posted 12 October 2004 - 10:26 PM

if the controls are working, but the tests aren't.... well at least your taq is still alive (do youkeep it on ice at all times?).
Have you tried varying the concentration of Mg ions in you mixture?
Have you tried altering the temperature slightly?
I'm thinking if the quality of the DNA you're using isn't good... is it a low concentration of DNA, or not purified, or just really fickle? Can you make more DNA?
Try getting someone else to make the PCR up for you, and see if it works in their hands.... if it does. :rolleyes: haha, it's their job now.... if it doesn't, well, at least it's not you, and then you can move on from there.
Sorry i'm asking more questions than giving answers for.
vetticus

#18 ocean

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Posted 12 October 2004 - 11:09 PM

hello,

thank you for your reply...yes i have tried different buffers and i'm using a touch down thermocycling conditions where the annealing temp is actually low than that described in the literature. i think our problem is our controls and samples are extracted from different sources and because of that they also have differently purity. the controls are much more pure. we are now going to reoptimise using samples, just to get the assay working. we have also decided to use DMSO 5%...but i think i can get away with 2%. i just have to keep trying :rolleyes:
i haven't got someone else to do it for me yeah :D

hope it works now.....

#19 ocean

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Posted 13 October 2004 - 11:46 PM

so far so good with the addition of DMSO :D




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