Notices
Results 1 to 10 of 10

Thread: PCR

  1. #1 PCR 
    Forum Bachelors Degree
    Join Date
    Jul 2008
    Posts
    420
    Hi,

    I'm having real trouble amplifying a gene of interest by PCR. I have tried it several times now, and have tried adjusting the annealing temperature. By agarose gel electrophoresis i get nothing (except the DNA ladder), except once at a reduced annealing temperature I got non-specific product bands.

    I'm beginning to think that maybe I am inadvertantly introducing pipetting errors. If pipetting errors are introduced into the PCR reaction, are they likely to cause the PCR reaction to fail? I guess that even a tiny pipetting error would make a big difference to the overall concentration in the reaction tubes.

    Also, does it matter in what order things are added to the reaction tubes? I have been doing this:

    Make a 5 x mix containing

    MilliQ H2O 152.5uL (to make it up to 50ul per reaction)
    Pfu buffer - 25uL at 10x
    Forward primer - 25uL at 10uM
    Reverse primer - 25uL at 10uM
    dNTPs - 12.5uL at 10mM

    (Added in this order)

    48uL of this 5 x mix was pipetted into each of 4 reaction tubes

    1uL template DNA was added per tube, followed finally by 1uL Pfu

    The reaction tubes were spun down in a microfuge at 4.5 krpm before being put into the PCR machine

    Please, if anyone could advise me as to what I am most likely to be doing wrong, I would be eternally grateful. I REALLY want this PCR to work!

    Is it most likely to be a pipetting error? The wrong order of adding things?

    Best wishes,

    Tridimity


    Reply With Quote  
     

  2.  
     

  3. #2  
    Forum Professor
    Join Date
    Sep 2007
    Posts
    1,079
    No, it will not be down to pipetting errors.

    Either your DNA is leading to problems (try using less DNA, diluted into TE, and run some of your DNA on a gel to gauge purity and integrity) or your primers or dNTPs are no good. Double check your primer sequences and try new aliquots, also try a fresh aliquot of dNTPs.

    Your order of addition to the master mix is fine.


    Reply With Quote  
     

  4. #3  
    Forum Bachelors Degree
    Join Date
    Jul 2008
    Posts
    420
    Thanks,

    I will try again
    Reply With Quote  
     

  5. #4  
    Forum Cosmic Wizard spuriousmonkey's Avatar
    Join Date
    May 2005
    Posts
    2,191
    what's your positive control?
    "Kill them all and let God sort them out."

    - Arnaud Amalric

    http://spuriousforums.com/index.php
    Reply With Quote  
     

  6. #5  
    Forum Bachelors Degree
    Join Date
    Jul 2008
    Posts
    420
    My positive control was TAB1 and primers, which worked recently on the same PCR programme for someone else in the Lab, so it is definitely something I personally am doing wrong

    Still don't know what. Depressing

    I have tried using fresh reagents, changing one at a time and then finally the whole lot

    I think I may have added the wrong volume of H2O to the 5 x mix.. I think I was 10uL out
    Reply With Quote  
     

  7. #6  
    Forum Professor
    Join Date
    Sep 2007
    Posts
    1,079
    Have you tried using less template. Do you know that your primers are perfect matches for the sample you are targeting?
    Reply With Quote  
     

  8. #7  
    Forum Bachelors Degree
    Join Date
    Jul 2008
    Posts
    420
    Yes, the primers are perfect
    Reply With Quote  
     

  9. #8  
    Forum Masters Degree samcdkey's Avatar
    Join Date
    Jun 2006
    Posts
    640
    Maybe there is a primer template mismatch or the CG content is too high. Try lowering the annealing temperature.
    Reply With Quote  
     

  10. #9  
    Forum Professor
    Join Date
    Sep 2007
    Posts
    1,079
    Well, that leaves your template as the most likely suspect. Depending on your extraction protocol, any number of compounds could prohibit amplification. Phenol and chloroform are two likely suspects, and provide excellent reasoning for including a positive control in every run. Depending on the source of your material (tissue, environmental sample, etc), other chemicals such as humic compounds, which copurify with DNA, can interfere with amplification.

    Any of these problems can be easily overcome by reducing the amount of template that you add to your reaction. Try a 1:10 dilution and a 1:40 dilution. Alternatively, dialyse your DNA sample to remove potential inhibitory compounds. A fresh extract is another possibility; be certain to remove all traces of phenol and chloroform or other organic solvents, which are excellent denaturants, in your next extraction.

    This of course is all a long-winded way of asking whether you have tried diluting your template.
    Reply With Quote  
     

  11. #10  
    Forum Cosmic Wizard spuriousmonkey's Avatar
    Join Date
    May 2005
    Posts
    2,191
    Quote Originally Posted by tridimity
    My positive control was TAB1 and primers, which worked recently on the same PCR programme for someone else in the Lab, so it is definitely something I personally am doing wrong

    Still don't know what. Depressing

    I have tried using fresh reagents, changing one at a time and then finally the whole lot

    I think I may have added the wrong volume of H2O to the 5 x mix.. I think I was 10uL out
    Let me rephrase the question.

    what is your positive control for your DNA sample then?
    "Kill them all and let God sort them out."

    - Arnaud Amalric

    http://spuriousforums.com/index.php
    Reply With Quote  
     

Bookmarks
Bookmarks
Posting Permissions
  • You may not post new threads
  • You may not post replies
  • You may not post attachments
  • You may not edit your posts
  •