18 replies to this topic

[ocean]

hello to anyone who has had this problem.

i have pcr products from a run of 6 to 7 samples and conducted RFLP and got the correct size products. but when i repeat the procedure for a larger sample..say 45 samples...it fails??

[blasko]

Did you cool your samples?
It's necessary...at big numbers of samlpe RFLP ..

[ocean]

blasko, on Sep 22 2004, 04:23 AM, said:

Did you cool your samples?
It's necessary...at big numbers of samlpe RFLP ..

helllo, yes i did.  even when i repeated some of the samples again (in small numbers), the pcr failed  

[ocean]

actually, what do you mean by cool down....holding?  if so for how long?  

[kant0008]

Hi,
seeing your PCR failed the second time round, even with the small number of samples, it sounds to me like soemthing in your reaction has gone off. If your other PCRs (if you are running any) work, and it's just this one, then your DNA is being degraded. otherwise it could be any of your reagents, dNTPs and Taq being the first ones to check.
Try running out your DNA to check integrity, or spec it for A260.280 ratio. Also change to fresh reagents.
Good luck!

[ocean]

kant0008, on Sep 22 2004, 05:09 PM, said:

Hi,
seeing your PCR failed the second time round, even with the small number of samples, it sounds to me like soemthing in your reaction has gone off. If your other PCRs (if you are running any) work, and it's just this one, then your DNA is being degraded. otherwise it could be any of your reagents, dNTPs and Taq being the first ones to check.
Try running out your DNA to check integrity, or spec it for A260.280 ratio. Also change to fresh reagents.
Good luck!

helllo..i have check the dNTP's and Taq...they appear fine as it's also worked on another PCR...i have also checked the quality of the DNA (quality a bit poor).  i'm currently running another PCR on another SNP...so i hope it works!

[ocean]

my last PCR failed again....  
wat can i do...how low can you use Taq once it has been opened.  the one i am currently using was opened in late July?

[kant0008]

Hi,
it shouldn't matter when you open your Taq, but rather how it is stored and manipulated (ie, at -20C, always on ice, etc).
Is ther a PCR which always works in your lab you could try?
Or maybe try another vial of Taq?

[ocean]

Hello, i'm just set up another pcr that worked...to check to see if it works.  i have a feeling it 's the primers? maybe fresh stock has to be made (was made not long ago   ).  thanks..i will keep trying

[ocean]

only the controls worked.....stuck!!!

[kant0008]

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)?

[ocean]

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.

[smesme]

Hi,

2 suggestions:  

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) ...

regards, Søren

Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark

www.ProbeLibrary.com
One Real-time PCR kit, which covers 38.565 genes

[ocean]

hello,  

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

[ocean]

hello everyone...
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..