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Troubleshoot PCR smear problem - all I got was smear!! (Aug/18/2004 )

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It seems that this topic has been answered to death, but I had a thought that no one else brought up, and it may help someone in the future?

Oligos will degrade over time, even when properly resuspended in nuclease free water or buffer and aliquoted and stored at -80...eventually they will degrade and this could cause non-specific priming. Based on my personal experiences in the lab, I would consider this problem first if I began to see smeary gels, particularly if I were careful about good technique everywhere, especially if the problem seems to get worse over time.

I had a similar experience once and spent a few months going through every detail of my protocol...remade and reordered my other solutions 8 trillion times and was very frustrated...and it just turned out to be oligo degradation.

-aimikins-

hi
do mean by oligonucleotides the primer stock prepared ???
and if that is the case how can u maaake sure that ur primer stock is dead???????

-lula-

Hello,

I hope everybody that encounters a problem with PCR is able to read this. I was trying to amplify a ~600 bp fragment previously amplified from Drosophila genomic DNA. So, the PCR using gDNA as template worked, but the PCR from ~600 bp fragment as template did not work. I tried everything from changing Ta, Taq, [MgCl], machines, new primers.....

Any ways, I diluted the template from 1:10 to 1:10,000. I observed the best results at a 1:100 dilution. So try this, see if it works...

Good Luck, Jorge.

P.S. If you want a picture of the gel email me.
munizjg@email.uc.edu

-Esloquiyao-

QUOTE (kudna @ Sep 22 2004, 08:24 PM)
Thank you for your replies.  It turns out the carry-over contamination may infact have been the problem.  After making up fresh solutions and ordering new primers, taq, dNTPS's etc, using a new set of pipets, and setting up post-PCR in a different lab (don't know which of these or how many of these solved the problem)  the streaking has disappeared and the bands are bright. 
The Mg2+ concentration I'm using is 1.6mM which I haven't changed so I don't think that affected it but thank you for the suggestion, and the thank you for the troubleshooting site recommendations Ihab.  Very helpful.



Kudna



could have been the primers, did you definitely order them correctly the first time, sounds like only one primer was binding, this gives a smear

My student before was notorius for ordering primers but forgetting to reverse compliment the reverse primer, i.e cutting and pasting the last 30bp to the stop codon, not rev comp ing it and ordering, so he got smears.

could be your primer company made a bloop too!

R

-Yeastman-

QUOTE (kudna @ Sep 22 2004, 02:24 PM)
Thank you for your replies.  It turns out the carry-over contamination may infact have been the problem.  After making up fresh solutions and ordering new primers, taq, dNTPS's etc, using a new set of pipets, and setting up post-PCR in a different lab (don't know which of these or how many of these solved the problem)  the streaking has disappeared and the bands are bright. 
The Mg2+ concentration I'm using is 1.6mM which I haven't changed so I don't think that affected it but thank you for the suggestion, and the thank you for the troubleshooting site recommendations Ihab.  Very helpful.



Kudna


might have been the dNTPs. Degradation of dNTPs can lead to smearing of pcr product. Ran across this problem while doing 16-23 rDNA sequencing.

-dobbiewalton-

We are currently experiencing similar problems with microsatellites in maize. We observe what appears to be a high molecular weight product apparently stuck in the wells (running on 4% SFR agarose gels) and a faint smear down the lane. This occurs regardless of our Taq source and the thermocycler used for the reactions. Tried and trusted protocols are used, so it is doubtful that the problem lies in them.

The obvious next steps will be taken; new DNA extraction, new reagents and buffers, etc. I am particularly curious as to the probable causes of this phenomenon.

-szalmasj-

Your streak problem may be due to contaminted combs. If the combs are not washed properly the agar dries on them and due to which wells are not formed properly. Check on them
Thanks
Solution

-Solution-

Combs are pristine-clean. Thinking it is a DNA issue. Going to try new DNA I extracted myself. Gotta love breaking in a new technician.



QUOTE (Solution @ May 20 2005, 06:22 PM)
Your streak problem may be due to contaminted combs. If the combs are not washed properly the agar dries on them and due to which wells are not formed properly. Check on them
Thanks
Solution

-szalmasj-

Hi Kudna,

congrats on getting your pcr to work finally... i've been in the molecular minefield for some time now (not on pcr but several other areas of cloning etc) so know how frustrating it can be...

as a newbie on this site (although not in the lab) i was impressed with the banter so thought i'd chip in....

w.r.t. mol biol work, the biggest contaminant i've come across is that of nucleases... a little contamination in any of your reaction components can be carried across to everything if the lab is blessed with an inexperienced student... then it can be the cause of many a smeary gel, inefficient digestions, incomplete ligations, etc etc etc....

and indeed often the best policy is to prep everything from scratch as one can spend so long trying to find the cause of an unaccountable smear, frustration becomes exponential with time!

all the best and thanks for the interesting read (procrastination i'm afraid!!)

globe trotter....

QUOTE (kudna @ Sep 22 2004, 08:24 PM)
Thank you for your replies.  It turns out the carry-over contamination may infact have been the problem.  After making up fresh solutions and ordering new primers, taq, dNTPS's etc, using a new set of pipets, and setting up post-PCR in a different lab (don't know which of these or how many of these solved the problem)  the streaking has disappeared and the bands are bright. 
The Mg2+ concentration I'm using is 1.6mM which I haven't changed so I don't think that affected it but thank you for the suggestion, and the thank you for the troubleshooting site recommendations Ihab.  Very helpful.



Kudna

-globe trotter-

Hi,
have you try a touch down PCR?
When my PCR give weak band I immediatly try a touch down PCR and to date it always has worked.
So maybe you can try it.
You can try this program:
3 min 94C
60s 94, 60s T(a)+2C, 60s 72C for 2 cycles
60s 94, 60s T(a)+1C, 60s 72C for 2 cycles
60s 94, 60s T(a), 60s 72C for 2 cycles
60s 94, 60s T(a)-1C, 60s 72C for 2 cycles
60s 94, 60s T(a)-2C, 60s 72C for 2 cycles
60s 94, 60s T(a)-3C, 60s 72C for 2 cycles
60s 94, 60s T(a)-4C, 60s 72C for 20 cycles
final extension 20min 72C

-delphine-

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