Im stuck with my PCR for nearly 2 months! - i've no bands!! (Oct/27/2005 )
[size=6]Hi all, im an intern attached to a research company, and im doing on a project and the lab work comprised of PCR and restriction digestion (RFLP)
In the begining, i used the first set of primers, i could get results almost every PCR i did, however, at the RFLP stage, my experiement failed, and there is no cut and bands appears on the same alightment as my undigested PCR product, thus my supervisor read up on another article and ordered a new set of primers. (2nd set)
From then, my PCR kept failing, there were times i could get the specific bands size of 158bp but the next time i do it again with the same PCR conditons, i got not get anything, and its just primer dimers at the bottom, and my supervisor commented that it could be that this 2nd set of primers were more sensitive and it could not tolerate any slight error, as compared to the first set of primers.
My PCR conditions were :
5ul of Promega buffer (without MgCl2)
1ul of 10mM dNTP
3ul of MgCl2
2.5ul of forward primer (10X Dilution--2ul of stock with 18ul of TE buffer)
2.5ul of reverse primer (10X Dilution--2ul of stock with 18ul of TE buffer)
2.5ul of 100% DMSO
31.5ul of dd water
1ul of Taq polymerase
1ul of gDNA (stock solution--undiluted)
Anealing temperature of 55 oC
I really do not know what is the problem behind my failed PCR, i have tried it for 20 over times, and sometimes i could get the bands sometimes i do not get, thus it could not be my reagents problem.
im really getting sad and depressed over this issue, and even my supervisor advised me to take a break off, but however i feel that thats an insultation to me as an intern and im worried that i could not finish my project in time as i only have 5 months and now i've already wasted 2 months for optimization.
i really need help... thanks...
Did you use filter tips for aliquoting the sample?
Are you sure the primer are thaw on ice?
Are you sure you are very careful of not cross- contaminate your primer?
Do you make up the master mix, aliquot into the tube, then add the sample?
You may wish to make your master mix in the hood.
You may miss some cruical steps when you do it alone.
Ask your supervisor to do it together with you. So that you know what is wrong.
If your supervisor got problem. Then I think you may wish to order the primer again.
I hope it may help.
Hi, over a year back was in a human genetics lab where it was day in and day out of PCR optimisation. Let me give you the tips my post doc got me started on.
first of all have all your consumables in small aliquots so you dont have to put them through repeated freeze thaw cycles. Also use the cleanest available water. We used double ionized ultrafiltered water. We stocked it in 1mL aliquots and used a fresh aliquot for each master mix.
the actual optimization process starts in steps . Template titration- using a range of template concentrations . or fixed concentration and a range of volumes.
Mg titrations. again fixed concentration different volume. Primer titration. At each step u fix the value from the previous step. The last step is the temperature titration where u set up ur PCR at a gradient of +- 5 degrees from the melting temp of ur primer set. A hot start can be the last resort. Am assuming you have already checked your primers for optimum GC content and lenghth and yadayada.
Hope this helps. some ppl recommend throwing in DMSO in the mix. I never had to resort to that so cant say much abt it. Hope you get your PCR working soon.
Barrier tips help. change tips as often as u need to. Double glove to be extra sure. thaw everything on ice. spin down everthing for proper mixing and correct concentrations. Try different PCR tubes.
maybe a different Taq. And if you feel you've made a mistake at a point . start over again. It'll save you time over the long run.
This is important so I''ll stress this because I see these stories all the time------
The key with any/all PCR reactions and downstream applications is to have a postive and negative control. There is no way you or any of us on this board can figure out what is wrong with your reaction if you do not do the controls.We can give out all sorts of standard PCR problem junk (eg change H2O and reagents), but it is meaningless unless you derive a system to figure out what is really going wrong here. In your case it maybe more difficult (and do not know what you are studying), but if you have controls sequences you can tell if the problem is with the PCR, restriction digest, or both. My advice to you and students who come to me with similar stories, is to tell them to come up with controls and come back to me with the problems once they do the controls.
IMHO, this is what separates good scientists from bad scientists and prevents wasting of students/interns lives for months at a time.
Why are you just staying with this one PCR-condition? Try what "sprats" has said. Also, the remark of "Tap14" is very important, how can people here help if you're not saying what controls you do, or if you are doing the PCR on the same DNA-samples (why not have a DNA isolation problem, are you sure your template is not damaged, or that you wasted it somewhere in the extraction process?)...