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PCR stopped working!!! HELP!!!


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12 replies to this topic

#1 mjolner

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Posted 29 July 2009 - 02:04 PM

i dont know what to do, i am so desperate!!! i have 2 months to turn in a bunch of results or else i wont get my maternity leave.

things were working just fine until one day, my pcr stopped working. ill brief my experiment:

part 1: using invitrogen one step do a first round to make cDNA and a first product with GSP (can not use random primers, they give many unspecific products in the final step)
part 2: using invitrogen platinum taq use the first as a template to make a final nested product with inner GSP

one fine day all the bands just disappeared! i have done the following:

- reextracted all my samples in case my template is messed up, this does not help
- changed all my reagents... zilch!
- tried using different primers as a positive control... and yes, they do work... under all the conditions and reagents ive tried.

so, how can my primers just "stop working" from one day to the next? i even tried having my primers made all over again and nothing! i dont understand.

can anybody please give me suggestions... as i said, i only have 2 months to finish a large bunch of samples, or else my boss will take away my maternity leave until i hand in results. "the reaction stopped working, ive tried everything" isnt an excuse..... please, please help!

#2 ivanbio

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Posted 29 July 2009 - 02:20 PM

Hi mjolner,

The fact that your control primers work is good. This tells you all your reagents, and samples, are in good working order.

It is not entirely uncommon for a set of primers to stop working all of a sudden. My recommendation is to check and re-check that you are using the correct primers. The ones you got re-synthesized may not be the correct ones. I am sure you are very organized and know for sure (100%) that the primers you re-ordered are the same ones that were working before. Yet, there is always a chance there is a mistake somewhere. My recommendation, based on your explanation, is to go back and confirm for the nth time that the primers you synthesized are truly the ones that used to work. Once you determine that they are the right sequence, order them from a different vendor (if you can). If this is not possible, order them again from the same vendor. It is not impossible for even the vendor to ship you primers that were not actually the ones you ordered (most vendors get tons of orders and switching orders in not impossible). I would even go as far (paranoid) as ordering a second set of primers you know work with your new order, that way you can check that the new synthesized primers come from a good batch.

Good luck

Ivan
Carlsbad, CA

#3 mjolner

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Posted 30 July 2009 - 05:46 AM

thanks for the uppers!

yes actually, i have already sent the primers to be remade and double checked the seqcuence. same ones that used to work, but brand new. neither the old ones nor the new ones will work now. other primers from the same company have always worked for us, i trust them....
;)
any more ideas? anyone! thx :lol:

#4 ram

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Posted 30 July 2009 - 07:23 AM

My comment can appear fullish, and you would have done this earlier also ...but I will suggest to re-check all your things, procedure, PCR machine, PCR program .....EVERTHING a to z once again..do it yourself and also get it done from your colleague/s .
If your reaction has worked earlier, it should work now also..If at all it doesn't, can cloning your first product and using the plasmid for the 2nd reaction would be the option???
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#5 perneseblue

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Posted 30 July 2009 - 08:02 AM

the template might have become degraded from nuclease contamination. This is one of the most common causes for a PCR to suddenly stop working.

If there is sufficient material, check your DNA template on a gel for any signs of degradation. You will see a long smear of low molecular weight DNA if the sample is degraded.

If degraded, reextract the DNA from fresh material.
May your PCR products be long, your protocols short and your boss on holiday

#6 mjolner

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Posted 30 July 2009 - 08:03 AM

yeah, ive double triple cuadruple checked everything and tried all combinations possible.... :)
what plasmid are you talking about? i dont use a plasmid for my cloning, what i do is nested pcr on rna samples......

#7 perneseblue

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Posted 30 July 2009 - 08:23 AM

um... have you tried using a different PCR machine? It might be in need of servicing,

Do you have a PCR reaction which you can run on your template DNA that would be certain to give you a positive reaction?
May your PCR products be long, your protocols short and your boss on holiday

#8 hanming86

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Posted 30 July 2009 - 08:14 PM

PCR is indeed a nasty business from time to time.

Try diluting or increasing the conc of your sample . and PCR sometimes to suffer from template batch variation. if you could find ur old sample , perhaps u wanna use that for the PCR.

In any case, PCR machine itself is quite a problem too. there's one time i have six similar tube of reaction ( aliquoted from a mastermix , 100% identical) but from the PCR result only one out of 6 works. could be temperature control problem. who knows.
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#9 ram

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Posted 01 August 2009 - 03:33 AM

If you carry wish to carry out PCR directly over earlier PCR product, this sometimes doesn't work. Instead if you clone your first PCR product in suitable vector and then carry out PCR over the recombinant plasmid, it gives better results. Can anybody comment on whether this would be true in case of mjolner?
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#10 mjolner

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Posted 03 August 2009 - 01:08 PM

thank you all for your suggestions... still havent been able to get things to work, so any more ideas are grateful, but it really helps knowing theres support out there... even if its strangers online... :P
most of the things i have already tried, but i will recheck.... and brainstorm more possible problems.... if u think of anything else PLEASE let me know!!!
if i get the damn thing to work ill let u all know how the hell i did it.... :lol:

thx!

#11 Adrian K

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Posted 06 August 2009 - 10:56 PM

thank you all for your suggestions... still havent been able to get things to work, so any more ideas are grateful, but it really helps knowing theres support out there... even if its strangers online... :)
most of the things i have already tried, but i will recheck.... and brainstorm more possible problems.... if u think of anything else PLEASE let me know!!!
if i get the damn thing to work ill let u all know how the hell i did it.... :P

thx!



Hi, try to check your PCR temperature on the thermocycler whether is it accurate. Double confirm whether any jokers had messed up to your thermocycles internal settings like ramp rate etc. (something like this happened to me before)

Also, have you changed your PCR tubes to other brand lately? It might have a slight effect on your temperature. Some low quality brand had bad heat conduction activity.

Worst come to worst: try use miliQ water or nuclease free water in your PCR.

Also, you are using RNA as starting material...could it be your culture mutated, wrong expression condition etc?

just my 2 cents.
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

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#12 mjolner

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Posted 14 August 2009 - 02:42 PM

hi there... i got a hold of positive control primers for another zone that were able to verify that my templates are working.
i changed to a different taq buffer and now i get smudges with my primers that USED to work until a few weeks ago (and the new ones i sent to be made in case they had "died"), but nice bands with the positive control primers.
what could this be?
;)

#13 Adrian K

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Posted 16 August 2009 - 09:06 AM

Dear mjolner

let me rephrase your condition
you are using RNA as starting material, convert to cDNA, done PCR. Used to be working.

1)now, same cDNA (from above), perform PCR: but not working; possible reason cDNA degraded. Your control is working probably your control is target a repetitive gene or some highly expressed gene in your genome.

2)if, not same cDNA as above, and you had re-extract fresh RNA from new sample, convert to cDNA: and done PCR but not working; check your experimental condition as your cells might not being induced to express the gene you want.

just some ideas that came across...

good luck,
Adrian.
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




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