Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Primers stopped working!?


  • Please log in to reply
6 replies to this topic

#1 hvangapandu

hvangapandu

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 11 January 2011 - 08:54 AM

Hello,

I've been genotyping mice tail DNA using a set of primers. They were working fine till last month. I was trying to genotype them last week and it did not work, I changed the dNTPs, made a new dilution of primers(10um) from the stock(100um, always stored at -20C), used nuclease free water. But I do not understand whats the problem. It could possibly be the primers. I do not see any bands on the gel. The PCR clearly didn't work. I ordered a new set of primers. My question is how can primers go bad all of a sudden? They are suspended in TE. Did this happen to anyone else?

Edited by hvangapandu, 11 January 2011 - 08:55 AM.


#2 ivanbio

ivanbio

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 116 posts
7
Neutral

Posted 11 January 2011 - 11:07 AM

Primers typically do not go bad all of a sudden, but they do have a shelf life. How long have you had these primers? Did you use the same primers from the -20oC every time? In other words, did you freeze and thaw the same tube multiple times? Primers can withstand freezing and thawing a few times, but after 4 or 5 times the primers will indeed be completely degraded and you'll get the result you got. The best way to get around this is to make different aliquots of your primers, store them at -20oC, and only use each tube 3 or 4 times.

Of course failed PCRs could be due to a number of factors, but at least in this case it seems like using new primers should solve your problem.

Good luck!

Ivan
Carlsbad, CA

#3 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 11 January 2011 - 05:38 PM

Primers do not go bad all in one go. In fact, I have not actually seen a case where the fault is the primers.
And from what you have written, you do have stock primer solution and a diluted working primer solution.

Usually when a PCR suddenly doesn't work it is the fault of the template. Then the dNTP, then the thermocycler....

I would suggest that you run the PCR on a sample of DNA that you know will give you a signal.
I have seen peltier of a thermocycler stop working once. You might want to note where on the heating block did you place your tubes. Find out if anybody complained about PCRs not working
May your PCR products be long, your protocols short and your boss on holiday

#4 UBClabbie

UBClabbie

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 65 posts
8
Neutral

Posted 12 January 2011 - 03:54 PM

Use a fresh batch of mastermix. Multiple freezing and thawing of mastermix is usually the cause of no signal over freeze-thaw of primers, as long as your primer stock is of high concentration. I keep my stocks at 100uM and haven't a had problem with freeze thaw.

#5 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 708 posts
28
Excellent

Posted 13 January 2011 - 09:31 AM

Primers do not go bad all in one go. In fact, I have not actually seen a case where the fault is the primers.
And from what you have written, you do have stock primer solution and a diluted working primer solution.

Usually when a PCR suddenly doesn't work it is the fault of the template. Then the dNTP, then the thermocycler....

I would suggest that you run the PCR on a sample of DNA that you know will give you a signal.
I have seen peltier of a thermocycler stop working once. You might want to note where on the heating block did you place your tubes. Find out if anybody complained about PCRs not working


I saw it twice... 2 of the Biorad Mycycler in my collaborative lab had stop working more than half year ago... the repair almost cost about purchasing a new machine.
Be sure to check the running report everytime after run.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#6 csmartinho

csmartinho

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 02 August 2011 - 04:57 AM

Hi, this is probably extremely late, however I can tell you that using TE can definitely be the problem with your pcr amplification. The EDTA used for the TE is many times inhibitor of DNA polymerase enzymes. I suggest you precipate your samples and dissolve in water or just tris solution.

Good luck,
ClŠudia

Hello,

I've been genotyping mice tail DNA using a set of primers. They were working fine till last month. I was trying to genotype them last week and it did not work, I changed the dNTPs, made a new dilution of primers(10um) from the stock(100um, always stored at -20C), used nuclease free water. But I do not understand whats the problem. It could possibly be the primers. I do not see any bands on the gel. The PCR clearly didn't work. I ordered a new set of primers. My question is how can primers go bad all of a sudden? They are suspended in TE. Did this happen to anyone else?



#7 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,466 posts
247
Excellent

Posted 02 August 2011 - 08:43 AM

I'm sorry, I think this is just a myth. Do the math, instead of repeating a rumor. Your PCR reaction has a final concentration of magnesium at about 2 mM. If you have your primers at 10 uM in TE, and your final primer concentration is at 0.5 uM, then you are doing a 20:1 dilution. TE has 1 mM EDTA, so the final EDTA concentration is 0.1 mM (0.05 mM from the addition of each of two primers). So, the final magnesium concentration ends up at 1.9 mM, rather than 2 mM. This is inconsequential.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.