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Smear in long distance PCR


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39 replies to this topic

#16 Adrian K

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Posted 08 March 2011 - 12:17 PM

It seems that no reduction in the volumen occurs throught the PCR reaction (but I haven't measured them). The PCR tubes are new and also autoclaved. I usually arrange a final PCR volumen of 25 ul and I forgot to mention that I've been performing the DNA elution from the kit with nuclease free water!


Try to run a comparison between autoclaved and un-autoclaved tubes. From my experience, my autoclaved PCR tubes tends to have the issue of leakage and cause volume reduction, and this is the reason I never use autoclave tubes anymore. Not sure whether is this the case for you.

Why not try to email invitrogen and see what their tech have to say?
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#17 xabiarias

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Posted 08 March 2011 - 12:28 PM

Pretty interesting not using autoclaved PCR tubes. I have to probe it. I actually talked to Invitrogen's tech and they provided me a new kit (in the beginning I thought that the problem was the polymerase!) :unsure:

#18 Adrian K

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Posted 08 March 2011 - 07:56 PM

My PCR tubes were certified 100% virgin polypropylene and are lot tested and certified to be free of RNase, DNase, and Human DNA .. :D
I'm using PCR Tube with Flat Cap 0.2ml, Labcon [3936-500] Price RM180 @ ~USD45 per 1000pcs.

I was wondering why would you have to autoclave your tubes? Can share?

You don't autoclave your filtered tips, do you?
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

#19 Ameya P

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Posted 08 March 2011 - 08:56 PM

Xabi,

I too use un-autoclaved tubes for PCR and haven't had any issues so far... Mine are Axygen (PCR-02-C).
I dont use autoclaved tips either. They have filters though.

Adrian,

I dont think autoclaving the tube might be a reason for the precipitate.

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#20 xabiarias

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Posted 09 March 2011 - 05:14 AM

I've obtained the precipitate again in conditions without DMSO and enhancer A (needed for the correct amplification). So we can rule them out. I've just prepared a PCR reaction with Taq Platinum Buffer plus Mg plus dNTPs instead of SequalPrep Buffer (and of course with DNA, primers, polymerase and water). We'll see if it works out!

#21 xabiarias

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Posted 09 March 2011 - 05:23 AM

I didn't see your last post because didn't realize that they were in the other page...sorry!

I use un-filtered autoclavated tips. I always did this way in the university with good results and now in the hospital. In the beginnig (with this long PCR) I had good results (without filtered tips)but suddenly the precipitation arise! :angry:

#22 almost a doctor

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Posted 09 March 2011 - 05:44 AM

Hi, I have a question and what might be a silly suggestion but here goes.

Question: do you run no-template controls? If so, do you get the precipitate in those ones too? If you do that will rule out your DNA being the cause. If you dont get the precipitate there, then I'll try to clean the DNA of any posible contaminants. Or maybe simply dilute it, from the gel in your first post I'd have said your template concentration is too high.

Suggestion: you could test each individual reagent for "contamination". Set different tubes in which you omit one reagent at a time, so 1. no dNTPs, 2. no MgCl, 3. no buffer, 4. no DNA..... and then run the PCR with the same amplification conditions you've been testing. You wont get PCR product (not for most of the conditions anyway), but if the precipitation is caused by some sort of contamination (ie salts), you might still get it and hopefully can figure out which reagent is causing it. Mind you, you might not get precipitate in any of your conditions, but that will at least tell you it is some by-product of the actual PCR reaction. For this one testing the water might be tricky, as no water is probably not a good idea, but if you have the chance of using water from different sources that will help too. Which also brings me to another question, what is your water source?

And, just thought of yet another question. Do you always get this precipitate for every PCR, or is just a set of primers and a particular template?

#23 Zach T

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Posted 09 March 2011 - 03:56 PM

Hey, sorry for the absence. The smearing in my reactions was mainly caused by using too much taq I believe. I have been trying to optimize the LA pcr and tested out different concentrations. .2ul of taq produced pretty good bands but i noticed an increase in smearing as i increased the concentration .5ul per rxn. hope this helps some, i'm still having issues with my rxns as well. logical troubleshooting just doesn't really seem to be working

#24 Ameya P

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Posted 09 March 2011 - 10:01 PM

Suggestion: you could test each individual reagent for "contamination". Set different tubes in which you omit one reagent at a time, so 1. no dNTPs, 2. no MgCl, 3. no buffer, 4. no DNA..... and then run the PCR with the same amplification conditions you've been testing. You wont get PCR product (not for most of the conditions anyway), but if the precipitation is caused by some sort of contamination (ie salts), you might still get it and hopefully can figure out which reagent is causing it. Mind you, you might not get precipitate in any of your conditions, but that will at least tell you it is some by-product of the actual PCR reaction. For this one testing the water might be tricky, as no water is probably not a good idea, but if you have the chance of using water from different sources that will help too. Which also brings me to another question, what is your water source?


Hey, I like this idea.

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#25 xabiarias

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Posted 11 March 2011 - 09:00 AM

Hi everyone,

Yesterday I suspected about the reaction buffer being the problem but now I have no doubts about it.

Here is a picture of my last PCR:

1-7: Taq platinum buffer + 0,5mM MgCl2 (final concentration)+ 0,8 mM dNTPs (final concentration) = A
8-12: SeqPrep buffer (MgCl2 and dNTPS incorporated in the kit)= B
1 and 13: A + polymerase
2: A + 30 ng DNA + polymerase
3: A + primers (about 1 uM final concentration)+ polymerase
4: A + 30 ng DNA + primers + polymerase
5: A + 30 ng DNA + primers + DMSO (7% final)+ polymerase
6: A + 30 ng DNA + primers + Enhancer (from the SeqPrep kit 1x final concentration)+ polymerase
7: A + 30 ng DNA + Primers + DMSO + Enhancer + polymerase
8: B + 30 ng DNA + Primers + polymerase
9: B + 30 ng DNA + Primers + DMSO + polymerase
10: B + 30 ng DNA + Primers + Enhancer + polymerase
11: B + 30 ng DNA + Primers + DMSO + Enhancer + polymerase
12: B + 30 ng DNA + Primers + dNTPs + polymerase
14: B + polymerase
15: newest Seqprep Buffer + polymerase

By simple observation of the PCR tubes after the PCR reaction I could easily note the precipitation on tubes 9,10 and 11.

The point is that the precipitation only appears when I combine the seqprep buffer with DNA or primers. Could it be posible that for some reason something is co-purifying along my DNA sample and only interfers with Seqprep buffer (and not with Taq Buffer) causing the precipitation? Or could it be posible that some contamination had happened in the Seqprep buffer (perhaps a funghi?) that only in the presence of DNA produces the precipitation? Remember that we use the same room for all the process and we do not use laminar flow cabinet.

I'm not sure if with Taq Platinum Buffer this is going to work (the amplification of my 12 kpb bands!) but al least I have no precipitation! :rolleyes:

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#26 Zach T

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Posted 11 March 2011 - 01:14 PM

Hmm, that's interesting that the buffer was giving you the precipitate. I think we are probably having different issues but with very similar results. b/c i've occasionally gotten great amplification, but in others had smearing on the high end (10kb+ to well) which was probably due to excess taq, but then I've also had smearing before on the lower end like your pic....so not really sure the issue in my rxns.

Glad you figured out the issue!

Edited by Zach T, 11 March 2011 - 01:15 PM.


#27 Ameya P

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Posted 11 March 2011 - 09:21 PM

9: B + 30 ng DNA + Primers + DMSO + polymerase
10: B + 30 ng DNA + Primers + Enhancer + polymerase
11: B + 30 ng DNA + Primers + DMSO + Enhancer + polymerase
12: B + 30 ng DNA + Primers + dNTPs + polymerase
By simple observation of the PCR tubes after the PCR reaction I could easily note the precipitation on tubes 9,10 and 11.


Xabi,

9, 10 and 11 give you precipitate, but 12 does not. So shouldn't it be the Enhancer or the DMSO along with primers and DNA the problem. Well, yes nothing happens with A + DNA+ pol+ primers, but you were not using that buffer before anyways...

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#28 perneseblue

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Posted 11 March 2011 - 11:21 PM

What is the DNA resuspended in?

I am wondering if the DNA had been resuspended in phosphate buffer.
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#29 xabiarias

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Posted 12 March 2011 - 12:42 AM

DNA is eluted in water nuclease free from Invitrogen.

#30 xabiarias

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Posted 15 March 2011 - 02:33 PM

Hi guys,

Here comes another pic:

1-5:seq prep buffer (MgCl2 and dNTPs incorporated in the kit)=B
6-10:taq polymerase buffer+MgCl2 (o.5 mM final concentration)+ dNTPs (0.8 mM final concentration)=A
1: B + polymerase
2: B + polymerase + DMSO final 7% + Enhancer A 1x final
3: B + polymerase + 30 ng DNA
4: B + polymerase + primers (1 uM final more or less)
5: B + polymerase + primers + DNA
6: A + polymerase + primers + DNA 30 ng + DMSO 7% final + Enhancer A 1x final
7: A + polymerase + primers + DNA 60 ng + DMSO 7% final + Enhancer A 1x final
8: A + polymerase + primers + DNA 90 ng + DMSO 7% final + Enhancer A 1x final
9: A + polymerase + primers + DNA 200 ng + DMSO 7% final + Enhancer A 1x final
10: A + polymerase + primers + DMSO 7% final + Enhancer A 1x

GT_ameya, I think that DMSO and the Enhancer are not producing the precipitation due to the condition 2 which is clean even in the agarose gel. However, something happens in tubes 3 and 4, 5 (the precipitation in tube 4 was clearly noted by simple visualiaztion) when DNA and/or primers are added. Despite failing to obtain the desired band (around 12 kpb) in 6-10 conditions, the gel and the tube visualization clearly indicates that no precipitation has ocurred again using the taq platinum buffer.

The question is, what is producing the precipitation? I have received a replied from Invitrogen telling me that buffer ingredients are confidential so not sure that I gonna make it with this Taq Platinum buffer. :blink: What do you think?

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