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PCR problem


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#1 Nathan

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Posted 29 January 2009 - 10:05 PM

hi am struck up with my PCR for the past one month . am getting a band in the control ;) i did the same PCR about 10 times but the result s same.. i wear gloves and all my equipments are fresh and new. i even changed my primers but am encountering the same problem. the primers that i have designed is conserved in all bacerial genomic DNA.. am suspecting that the Taq polymerase might have a small amount of Taq genomic DNA which is getting amplified.. how can i get rid of this contamination.. am using Taq pol and buffer from NEB, all other PCR reagents from invtrogen.. help me pleasssseeee.. am frustrated to the core.... waiting for your replies

Edited by Nathan, 30 January 2009 - 03:16 AM.


#2 T C

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Posted 30 January 2009 - 12:51 AM

Hello

I don't really understand by what you mean by getting a band in the template?

It is clear that you don't get yr desired amplification, but what do you see in the gel? Its a good idea abt to write something about what you are doing....will become easy to troubleshoot. ;)

TC

#3 Nathan

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Posted 30 January 2009 - 03:14 AM

Hello

I don't really understand by what you mean by getting a band in the template?

It is clear that you don't get yr desired amplification, but what do you see in the gel? Its a good idea abt to write something about what you are doing....will become easy to troubleshoot. :)

TC

hi thanks for replying i meant that am getting a band in the control.. actually am trying to amplify a gene which is present in all bacteria. am amplifying that gene from leptospira spp.. my primers are conserved in all bacterial species. so i guess the taq pol enzyme may contain a small amount of taq genomic DNA which is getting amplified in the control.. is my point right? how can i get rid of this problem.. ;) please reply ASAP help me

#4 merlav

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Posted 30 January 2009 - 03:44 AM

Change all reagents!!Begin with a new water, buffer and new dilution of the primers. If still got contamination use a new stock of the Taq. and if you still got contamination in the negative control you will need a new batch of primers.
Try to do the reactions in a PCR workstation or biological hood, Clean it with DNAzap or similar detergent. UV the gloves, tubes, water, tips, pipets and racks for at least 30 min. Change tips between reagents and samples. If tips touch any surface discard it. If possible use tips with filter. I know they are a little bit pricier, but use them only for PCR. Have your own tips and if possible your own working solutions.
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#5 T C

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Posted 30 January 2009 - 03:52 AM

Hey

To check that possibility, check the sequence of your gene in the genome of the organism from which taq pol has been isolated. Your primers should also work with this sequence. However, I doubt if this would be the case as I expect changes at the nucleotide level. Also, the commercially available enzymes are expected to be of high purity.

Another possibility is that something is wrong with the reagents that you are using. Water?

What all do you have in the control and how is it different from yr test reaction?

TC

#6 mdfenko

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Posted 30 January 2009 - 07:21 AM

if you think it is the taq then try changing to a different taq or different polymerase.
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#7 Unagi

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Posted 30 January 2009 - 02:46 PM

I'm assuming you are having trouble with your neg. control? What is your PCR target? If it's the 16s gene, then you might have ongoing troubles.
Like previous posts have already stated, having your own reagent stocks and tips is important, and UVing your gloves, pipettes, tubes and tips can help. Also work in a UV equipped laminar flow hood (uv it before using) Filtered tips are a must in my opinion. The negligible price difference can make a world of difference.

Most POL enzymes will have trace amounts of 16s bacterial DNA, not from Taq, but from the bacterial cultures which are used to commercially express them. There are some polymerases available commercially which have stated low levels of bacterial DNA. You might want to try those.

What kind of water are you using? I would reccomend presealded PCR grade water or water for injection (human use). If you want to do a bit more work, you could even try to DNAse your water (but remember to denature the water-enzyme mix before applying it to your PCR mix).

Chances are, that even after all of that, you will still get some level of residual non-specific template there. The good news is that if your specific template is there, you should see a obvious difference between band intensity between your neg control and sample. Of course, then you still would have to sequence it to confirm the identity of the amplicon.

All this of course is assuming that your primers are specific (well, as specific as a 16s PCR can be). Make sure that your primers are as different from all the other available bacterial sequences available on Genbank as possible (especially their 3' end), while sill being conserved in the Leptos.

#8 Nathan

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Posted 31 January 2009 - 01:57 AM

I'm assuming you are having trouble with your neg. control? What is your PCR target? If it's the 16s gene, then you might have ongoing troubles.
Like previous posts have already stated, having your own reagent stocks and tips is important, and UVing your gloves, pipettes, tubes and tips can help. Also work in a UV equipped laminar flow hood (uv it before using) Filtered tips are a must in my opinion. The negligible price difference can make a world of difference.

Most POL enzymes will have trace amounts of 16s bacterial DNA, not from Taq, but from the bacterial cultures which are used to commercially express them. There are some polymerases available commercially which have stated low levels of bacterial DNA. You might want to try those.

What kind of water are you using? I would reccomend presealded PCR grade water or water for injection (human use). If you want to do a bit more work, you could even try to DNAse your water (but remember to denature the water-enzyme mix before applying it to your PCR mix).

Chances are, that even after all of that, you will still get some level of residual non-specific template there. The good news is that if your specific template is there, you should see a obvious difference between band intensity between your neg control and sample. Of course, then you still would have to sequence it to confirm the identity of the amplicon.

All this of course is assuming that your primers are specific (well, as specific as a 16s PCR can be). Make sure that your primers are as different from all the other available bacterial sequences available on Genbank as possible (especially their 3' end), while sill being conserved in the Leptos.


thanks for ur suggestion guys.. i have ordered for new taq polymerase and i would be getting it tomo.. hope everything goes alright.. :lol:

#9 Nathan

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Posted 31 January 2009 - 02:09 AM

Hey

To check that possibility, check the sequence of your gene in the genome of the organism from which taq pol has been isolated. Your primers should also work with this sequence. However, I doubt if this would be the case as I expect changes at the nucleotide level. Also, the commercially available enzymes are expected to be of high purity.

Another possibility is that something is wrong with the reagents that you are using. Water?

What all do you have in the control and how is it different from yr test reaction?

TC


am using nanopure water.. in test i have all the reagents + the template DNA.. my negative control contains all reagents except template...

#10 T C

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Posted 31 January 2009 - 02:54 AM

Hey

Try changing your water, maybe the stock is contaminated. I generally use normal MQ water, freshly autoclave it and syringe filter it with 0.22 micron filter while the water is still warm and store it in aliquots sealed with parafilm. These aliquits I use and throw.

Alternatively, give the DNA and oligos to some friend of your in another lab (if the person is willing). This will tell you if there is something wrong with any of the lab stock.


Also, it is possible that someone used a contaminated tip to use the enzyme and so you are taking template + enzyme from the enzyme vial. It happened in my lab once, when a short term trainee used a contaminated tip.

TC




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