Yet another PCR problem/question - (Sep/20/2006 )
Hello,
I took over a position three months ago that requires a lot of genotyping mice by PCR. The lady who I took the position from has notebooks and notebooks full of successful PCR pictures. I am using the same primers, same thermocycler conditions, same pipettes as she used and I am not getting any bands at all. I have a bright "band" remaining in the well with a streak down the lane in some, and in others I am not getting anything at all on the gel except for the ladder lane. I have tried the PCR on at least five different colonies with the same negative results. My water is not contaminated, I have reordered and rediluted primers, remade working primer solutions, ordered new taq and mastermixes . . . I just don't understand.
I have even tried the PCR on already confirmed animals' stored DNA in the -20 that she had used. I don't have any results there, either.
What could I be doing so differently than she did? I am fairly experienced with PCR and sterile technique, etc. Please, any and all suggestions will help!!
Thank you!
I would guess it's your gel, but I'm no expert in PCR.
How do you think it would be my gel? Do you think maybe the agarose is bad? I just use 1.5% and 2% agarose/TAE gels with ethidium bromide . . .
Do you make it fresh right before pouring the gel? The band staying at the top of the gel in the well is what makes me think it's your gel. How big is your product supposed to be?
Some examples of the products I should be getting are: 676, 366, 500, and 150. I do make a fresh gel before I run one. And now since I'm having so many problems, I am changing the buffer in the gel box and washing it out before I run the gels in it. I'm trying to be as clean as possible, but to no avail.
. . . By the way, thank you for your help and suggestions so far!!
No problem! I'm bored at work...how about your sample buffer? I don't have a recipe because I don't work with DNA anymore, sorry. That's all I can think of.
Hmm, maybe I'll start with a fresh batch to see if that makes a difference. My fingers are crossed!
Thanks!!
I find debugging such a problem easier when I look back at the basics of PCR.
THe PCR reaction mix
- Buffer
- dNTP
- Template
- Primers
So first crime suspect is the template DNA.
First question... is there any DNA in the tube? Or has it all degraded while in storage. Confirm this by gel. Run a raw sample. Cut a second sample with restriction to show that it can be cut.
Next DNA is usually suspended in TE, the EDTA is a big problem. Did the lady have a 'special' DNA storage solution like something enriched with EDTA and spiked with glycerol? And for some species, even 'clean' DNA will not amplify well, some kind of contaminant gets through despite all said and done.
Solution; Dilute the genomic DNA sample. Do a titration experiment 1/5, 1/10, 1/20. 1/50, 1/100 dilution.
My genomic DNA actually gives a better signal at 1/100 dilution.
Hope this helps.
It is always the DNA template, that is the rule.
It could be something really simple. My student was failing to use the hot lid on the cycler, for example, and got no results.
hello
wat i feel the prob may be with ur template DNA..how long has it been since ur DNA sample has been isolated..if its a pretty old sample,then do check whether it is still intact or getting degraded..also try varying the quantity of DNA to be used,along with try preparing the PCR mix with varying concentrations of reagents..if u try 5 to 6 different concentrations,any one is sure to work out..and also try bringing out minute changes in ur annealing temperatures..recheck the Tm(melting temperature) and den run another set of cycles..hope u'l get ur amplifications soon
good luck
anupama