PCR failing - (Sep/22/2004 )
Hi, you said before that your DNA quality is a bot poor, what are your readings or what does it look like? If you say your controls work, then probably the DNA is the problem. Is this genomic DNA? cDNA? Can you do another PCR on it that normally works (liek a housekeeping gene for RT_PCR)?
hello, the dna is genomic. the problem is the control were extracted from whole blood and the samples i am dealing with is from whole blood extracted from dried blood spots using qiagen. i am currently running a pcr on samples extracted by a college who extracted them from blood spots. i have compared dna quality with the other college using spectro and the purity is about the sample and even the concentration..si if this pcr works then it still doesn't help. the 260/280nm purity ranges from 1.088 to 1.99.
First of all primers are often the problem. Plain DNA primers are quite stable in high concentration , but once they are diluted, the can become quite unstable. Adding in a few freeze-thaw cycles doen't help. Try another stock dilution or order new ones, primers are cheaper than time.... Actually, if you analyse the primers its rarely over 80% that are full length, the rest are shorter synthesis products from incomplete synthesis.
Another factor which sometimes plays in is pipetting errors: when you mix a small mastermix, it is quite impossible to aliqute such small amounts of enzyme because of the glycerol, hence the smaller the reaction mix, the more enzyme is added, inadvertedly, which in some cases explains failures to scale up.
And use DNAse free water (autoclaved) ...
Søren M. Echwald, MSc., Ph.D.
One Real-time PCR kit, which covers 38.565 genes
thanks for advise...my next step will be to make up a fresh stock of primers if my next run fails. my problem is not only with one pair of primers...three of them are failing...but i think they were all ordered together
i have changed to a new vial of Taq polymerase and so far it's working..conducting another PCR and hopefully that works as well..
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?
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. 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.
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
i haven't got someone else to do it for me yeah
hope it works now.....
so far so good with the addition of DMSO