I did PCR with templates (same concentration for all templates) following this:
1. Negative control (Water)
2. pNL4-3
3. Negative control (TE)
4. pcDNA3.1
5. DuoFluo
I used the 1st PCR products for templates in the 2nd PCR. Primers were used in the 1st PCR and the 2nd PCR were the same. Both rounds had the same protocol.
After I had results (see picture below), I have 2 ideas.
1. Template 4 does not anneal with primers. And what I have in the 2nd PCR is contamination because I have no band in lane 4 in the 1st PCR.
2. Template 4 can anneal with primers, but after 2 rounds of PCR, it can be shown up. But what I am considering is all templates have the same concentration, if it does, lane 4 should have a band in the 1st PCR like others. I also checked the annealing capacity of primers and template 4, and they do not match.
Hence, can I conclude that lane 4 has contamination?
Hi,
What is the expected size of your amplification product in lane 4? Contrary to your assumption that it is a contamination, It is also possible that in your first PCR there was amplification in the later stages of the PCR cycle and the amount is too low to be detected on a gel (could be due to the template DNA - genomic/plasmid). For the second PCR, you are using a shorter linear fragment and the conditions probably worked well for its amplification!!! Remember PCR is very sensitive!!!
Hello, Kiran. Thank you for your comment!
I expect that lane 4 has no band. I think plasmid pcDNA3.1 does not anneal with my primers which is specific for HIV. However, if plasmid pcDNA3.1 which has the same concentration with other templates anneals with primers, will it show up in the 1st PCR?
Hello Trang,
Does that mean you were using lane 4 as a negative control? are you trying to say that the primers are not specific for pcDNA3.1?
It is not about having a band in lane 4 in the first PCR. Were the primers designed to amplify gene/fragment in pcDNA3.1?
Dear Kiran,
Yes, I mean I used pcDNA3.1 being negative DNA control. The primers I used are specific for two other templates (HIV). After I got results, my supervisors thought probably primers can anneal with pcDNA3.1. Therefore, I do experiments again today to check whether the band is contamination or PCR product.
You are getting a different size product in Lane 4. BLAST your primers against your pcDNA3.1 plasmid to see if they do match. Or you just have contamination. In that case, toss out ALL of your reagents (water, buffer, primers, enzyme, plasmids, TE), clean your work space and tools and try again.
Hello, Katie! Thank you for your comment. I did search in BLAST and they do not match. Absolutely, I get contamination. I threw all reagents and tried again, but I still had contamination only in pcDNA3.1, no contamination in water or TE. And my contamination band has the same size (1500bp) in all experiments. What should I do now? I think it may be due to my pipetting skills, but I don't know how to solve this problem.
Hi Trang, if you think it is a contamination due to your pipettes, I would suggest you to use filter tips. You mentioned that you changed all your reagents, have you changed Taq and primers as well?
Hi Trang, I would suggest you to do a repeat PCR with a new Taq pol! It is possible that your Taq is contaminated!
Thank you for your suggestion, Kiran! However, I consider that if Taq is contaminated, it should be a band in water or TE. Do you think so?
If your Taq is contaminated with pcDNA3.1 specific primers, you will not see a band in your negative control (no template available for amplification).
I am sorry, Kiran. Can you explain clearer? I cannot get your meaning.
Did you use the same set of primers for both your negative control and pcDNA3.1? If your taq pol is contaminated (during pipetting) with primers for pcDNA3.1, you can get amplification only in pcDNA3.1. Since your negative control has no template, just the primers will result in no amplification!
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