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weird PCR ask for help


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12 replies to this topic

#1 nccutudou

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Posted 17 January 2010 - 12:40 PM

Hi there, has anyone here encountered this problem: I did three batches of pcr last Monday and Tuesday. Target bands showed in gel. But when I run my latest PCR products (last Thursday and Friday), pcr product seems to get stuck in the well. I use the exactly same program and reaction system which worked before. I thought it may be the problem of reagents and I changed new tubes of buffer, dntp, primers and taq. It didn't work. I even used the exactly same templates which worked in Monday and Tuesday and the result are same. No target bands at all and products stay in gel wells. The taq i use is only 0.15ul vs 25ul. So it could not be the reason of excessive taq. what could be the reason? Thanks very much.
The attached is gel picutre. The left side are the targe bands (are boxed; 160bp) and the right side is latest gel result.

Attached Thumbnails

  • band.jpg


#2 claritylight

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Posted 17 January 2010 - 01:41 PM

It could be your gel. Sometimes if you don't clean out the wells of the gel, your DNA won't run properly due to obstructions in the gel itself. I would make another gel, wet it in the running buffer, then aspirate up and down with a pipette tip each well to clean it out. This way you are sure not to have obstructions. Or buy a pre-cast gel and run your material, and use that to compare. I bet it will run down as normal as before.

#3 nightingale

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Posted 17 January 2010 - 02:00 PM

I even used the exactly same templates which worked in Monday and Tuesday and the result are same.


what do u kindly mean by this ?

have you tried diluting the samples ?
" The more you learn, the more you realize how little you know ... "

#4 nccutudou

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Posted 17 January 2010 - 03:45 PM

It could be your gel. Sometimes if you don't clean out the wells of the gel, your DNA won't run properly due to obstructions in the gel itself. I would make another gel, wet it in the running buffer, then aspirate up and down with a pipette tip each well to clean it out. This way you are sure not to have obstructions. Or buy a pre-cast gel and run your material, and use that to compare. I bet it will run down as normal as before.


Thank you. i just have a question: if the problem is gel, why the marker can run down normally? The gel in picture was not the only one which had the problem. I run four gels last week. All the result were same but the marker was ok.

#5 nccutudou

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Posted 17 January 2010 - 04:00 PM

I even used the exactly same templates which worked in Monday and Tuesday and the result are same.


what do u kindly mean by this ?



have you tried diluting the samples ?





Thanks for your response. I doubted it could be the problem of template cause I had already changed all other reagents (dntp,primer,buffer and taq). So I tried the samples which I got bands before. To see if I can get bands or not. If yes, the problem is the templates I used later. But the answer is no. I got nothing with the templates that I used before.

I don't do this yet but the concentration of samples is not high.

Edited by nccutudou, 17 January 2010 - 04:04 PM.


#6 Adrian K

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Posted 17 January 2010 - 06:11 PM

Frankly, it does looks weird to me.
Just want to find out, what is your agarose gel percentage? Sometimes when increase the taq concentration, you will get a very high band (but very less likely you encounter it).
Make sure you are running your gel in your running buffer than pure water. Another possibility is might be your gel loading dye. I guess there might be something there which "stuck" your DNA there (too much glycerol??).

I really got no idea... just to voice out the thought from my mind and see whether it could help you in any way...
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

#7 nccutudou

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Posted 17 January 2010 - 08:12 PM

Frankly, it does looks weird to me.
Just want to find out, what is your agarose gel percentage? Sometimes when increase the taq concentration, you will get a very high band (but very less likely you encounter it).
Make sure you are running your gel in your running buffer than pure water. Another possibility is might be your gel loading dye. I guess there might be something there which "stuck" your DNA there (too much glycerol??).

I really got no idea... just to voice out the thought from my mind and see whether it could help you in any way...
Adrian


Thanks Adrian. I use 1% agarose gel to run product. I'm running buffer not water cause the marker run down normally. So does loading dye.

#8 Maddie

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Posted 18 January 2010 - 09:37 AM

I even used the exactly same templates which worked in Monday and Tuesday and the result are same. No target bands at all and products stay in gel wells.


This is weird indeed.

I want to make sure I understand. You took the PCR product from Monday/Tuesday, re-ran it on Thur and had DNA stuck in wells when before it had migrated?
Are you saying that the exact same PCR product is running differentely?
What's the size of the amplicon? Couldn't it be that last week was the weird gel?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#9 nightingale

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Posted 18 January 2010 - 09:55 AM

u r most welcome :D

are you saying that :-
u ran 2 PCRs for the same samples, the first gave bands while the other no ????

have you used the same cycler ?
" The more you learn, the more you realize how little you know ... "

#10 nccutudou

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Posted 18 January 2010 - 02:08 PM

u r most welcome :D

are you saying that :-
u ran 2 PCRs for the same samples, the first gave bands while the other no ????

have you used the same cycler ?




Thanks for your kindness, nightingale.

Yes, that's what I mean.

I've tried both same and different cyclers (Eppendorf Mastercycler Thermal Cycler). They gave same results.

#11 Adrian K

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Posted 18 January 2010 - 09:35 PM

Perhaps you can try dilute your PCR products (thursday and friday) and try run the gel again with the old product (monday and tuesday) on gel and see whats happen. For the time being, I can't really think of any possible reason for this situation as this never happen to me before.
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

#12 nightingale

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Posted 19 January 2010 - 01:50 PM

have u stored ur samples properly after running the first PCR ???
" The more you learn, the more you realize how little you know ... "

#13 Altair

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Posted 07 March 2012 - 03:19 PM

@nccutudou: were you able to figure out this problem? haha it seems I'm experiencing the same PCR issue as you =(.




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