Problem in negative control

Nov 16, 2015
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#1
Dear All,



The reason for writing to you is that I am facing one weird problem in my PCR assay. I am amplifying one gene which is around 1 KB in size for identifying any mutation in patient samples. This gene has high GC content and it has four primer set to amplify the entire gene.



I have successfully satndardized the PCR using Q-solution from qiagen. Last few days I am getting a faint band in the negative control (water). I assumed that it was due to contamination, hence I changed the entire reagents including the taq pol. However even with the fresh reagents and primers I could see faint band in my NC. In order to further trouble shoot, I ordered new primer sets and changed the even the PCR setting area into another room where this assay was never performed.



After doing the PCR even with new sets of primer in a new working room I can still see the faint band in my NC. I am unable to understand the reason for this problem. Would you kindly help me and suggest me what should I do in order to get rid of the contamination??



The primers which I am using has been taken from published literature and there are plenty of literature available using the same primer sets.



Looking forward to hear from you at the earliest. Kindly send me yr reply at firoz.ahmad@srl.in and cc to firozsrl@gmail.com since sometime scientist solutions is blocked in my organization due to firewall issues.



Thank you



Best regards



Firoz,

Mumbai- India
 

gilldean

New member
Nov 16, 2015
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#2
Hi Firoz.

It sounds like you did a lot of checking already, but here are 2 suggestions:

1. if you are having problems with contamination, use filter tips to keep the samples cleaner.
2. if you have a very effective PCR with a lot of product, spilling even a small amount from a well with product to a well without product when you load your gel can produce this effect. try loading the negative several blank wells away from the positive samples to test this, or load the negative on another gel. use a new tip for loading the negative (I don't know if you use the same tip for all loading or exactly what you do when you add load dye etc).
 

Ivan

Administrator
Staff member
Nov 16, 2015
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#3
Hi Firoz,



I agree with gilldean, you may want to consider filter tips or loading samples with a well in between to make sure you are not getting spill over.



Alternatively here are other things you may want to consider:



1. Your primers may not be of high enough quality. Unless you are 100% sure that your primers were synthesized properly, you may have primers that are of multiple sizes (a not uncommon result of primer synthesis), which can lead to the appearance of ghost bands that may resemble what you are getting. Get a new set of primers.



2. Run a second PCR reaction, with its one NTC, to see if the "contamination" issue occur no matter what PCR reaction you are running, or if it it specific to this particular PCR reaction. If you get bands even in NTC from other PCR reactions then you can be sure it is true contamination.



3. The quality of your DNA can have a significant effect on what gets amplified. If you use too much DNA you can easily expect to get extra bands that look like contamination. You may want to make sure your DNA is of high quality and do not use too much for PCR. Typically 100 nanograms of genomic DNA is more than enough.



Hope this helps. Good luck!




 
Nov 16, 2015
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#4
Hi Ivan and Gilldean..

Thank you very much for your views and comments...Yes I agree with to use and check with filter tips and I will do so today with one of my sample. However I have tried loading the NTC few wells away as well as in a new gel but the results were the same.



1) As far as the quality of the primers are concerned, I am very sure that they are of good qualtity and they were synthesized properly. In fact as I mentioned in my post, I ordered a complete new sets of primers and did the PCR with all the fresh reagents in a new working area but still I could see a few bands. What I am thinking now to reduce the number of PCR cycles from 35 to 30. What is your opinion about this strategy?? Will I be compromising with my result? considering my sampes are cancerous?



2) I have even tried this approach also and with second PCR reaction there is band in NTC



3) The DNA quantity is around 100 ng based on spec OD at 260 nm. However I would like to understand from you about how too much DNA can get extra bands that look like contamination in the NTC since we dont use any template in NTC?



Many thanks for all your comments.... I really appreciate it



Cheers :)

Firoz

 

Ivan

Administrator
Staff member
Nov 16, 2015
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#5
Hi Firoz,



Sounds like you have taken care of some of the possible items that could be causing you problems.



Here are responses to your questions/comments:



1. You can reduce your PCR cycles from 35 to 30. In the end, what is important is to consider how much background you are getting. If you can detect a signal in a NTC within ~3.3 PCR cycles from your positive signals then you can assume that about 10% of your regular signal is non-specific. If on the other hand you only detect a signal in your NTC within ~6.6 PCR cycles of your positive signals then only 1% of your regular signal is non-specific (which is statistically insignificant).



2. If we assume that your band in your NTC is indeed contamination then it does not matter what assay you use; you need clean your contamination. On the other hand is the band is coming from the assay itself, even in the absence of DNA, then designing additional assays could solve this problem.



3. Simple: the more gDNA you use in your positive samples, the greater chance you will get enough cross-contamination into your NTC to get a positive signal (which could be what you are getting). DNA can easily aerosol and contaminate multiple tubes when you are setting up your PCRs.



Cheers












 
Nov 16, 2015
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#6
Thanks a lot Ivan for sharing your views... I am trying some more experiments.... hence I will keep you posted about the progress..

Cheers :)