Today, I set up the 1st PCR with 2.0µl templates following:
1, 5: negative control (water)
2, 6: Plasmid 1 (0.2ng/µl)
3, 7: Plasmid 2 (0.2ng/µl)
4, 8: Plasmid 3 (0.2ng/µl)
Primers: gag-691F and HXB2-1759M, each one is 0.6µl.
Taq: 0.2µl
2.5mM dNTPs (1.6µl) diluted from 10mM dNTPs
25mM MgCl2 (1.0µl)
5x GoTaq Green Buffer (4.0µl)
H20 (10.0µl)
PCR reaction:
Initial denaturation: 95oC (2min)
Denaturation: 95oC (30s)
Annealing: 52oC (30s)
Extension: 72oC (1min10s)
Accumulating products: 72oC (5min)
Preservation: 4oC
Cycles: 30 times
Results and questions:
My 1st PCR products shown on the gel is Fig 1.
Then I used the 1st PCR products being templates for the 2nd PCR, respectively. Other reagents, PCR reaction are the same as the 1st PCR's. Then I got the 2nd PCR products in the Fig 2. But lane 2 (negative control) has a fuzzy band, lane 4 has a nonspecific band, and they are different from the 2nd set.
After that, I set up the 2nd PCR from the 1st PCR products in tube 1, 3 and 7 with the same reagents and PCR reaction, and do 2 sets. And the result is in the Fig 3, but the result is still inconsistent with others.
(I used 1kb DNA marker to run PCR products on the gel)
Can you help me to explain this problem and how can I solve it? Thank you so much!
Dear Trang,
First of all, you should not use the PCR product of your negative control form PCR1 in PCR2. That is not a negative control anymore. Specially after 60 rounds of amplification, you are to have non-specific bands.
Second of all, the a amount of PCR1 product used in PCR2 should be much lower, since you have lots of amplicon from your PCR1.
Third, did you use PCR1 products in PCR2 immediately after PCR1 was finished? If that is the case, I would cool down the PCR1 tubes for some time at 4C, just to make sure there are no aerosols going to your negative controls as cross-contamination.
Good luck,
Ali
Well, I didn't do that. I set up the 2nd PCR right after finishing PCR reaction of the 1st PCR.
Dear Trang,
First of all, you should not use the PCR product of your negative control form PCR1 in PCR2. That is not a negative control anymore. Specially after 60 rounds of amplification, you are to have non-specific bands.
Second of all, the a amount of PCR1 product used in PCR2 should be much lower, since you have lots of amplicon from your PCR1.
Third, did you use PCR1 products in PCR2 immediately after PCR1 was finished? If that is the case, I would cool down the PCR1 tubes for some time at 4C, just to make sure there are no aerosols going to your negative controls as cross-contamination.
Good luck,
Ali
When we are re amplifying PCR products, the amplicon is already saturated so we should dilute the PCR products to avoid non specificity. Have you diluted your 1st PCR products ?
The fluorescence reporter used may not be giving accurate efficiency of the PCR product
A. Dear Ali,
Thank you so much for your comments!
1. I am being trained, so that is my exercise I was taken.
2. I used BioRad thermocycler to run PCR, after finishing 30 cycles, it is automatically cooled down at 4oC.
Below is the picture showing the 1st time I did with the same reagents and protocol, but my teacher thought the 1st and the 2nd did not match, then I have to repeat some more times.
B. Dear Pratibha,
I have not diluted my 1st PCR products.
C. Dear Thimmaiah,
I have no idea about the fluorescence reporter, but I think it would give the right results.
I am wondering if your second set of primers can bind non-specifically to the template DNA used in your PCR#1. If I am understanding your PCR design correctly, it seems that the idea was to use only the product of the first reaction in your second PCR; if this was the case, the primers for PCR #2 may not have been checked against the template from PCR #1 which could explain the non-specific products you are mentioning. You can rule this out easily with a simple primer BLAST and/or by gel purifying the band you are interested in from PCR#1 prior to running your second reaction. As far as the negative control giving a band, have you checked the primer sequences (using a primer analysis software) for the possibility that your primers can anneal to each other and be extended by the polymerase? I have heard of this happening in negative controls occasionally, where the absence of a template DNA makes it more likely that primer-primer annealing will occur. Also are you standardizing the quantity of DNA you add each time? If in your second attempt at PCR #1 you got better amplification, but added the same number of uL of DNA to PCR #2, this higher template DNA concentration would make it more likely for there to be non-specific primer annealing to the template and could explain why you are now seeing these extra/non-specific bands.
Dear Trang, I agree with Hollie Rowlands answer:
" If in your second attempt at PCR #1 you got better amplification, but added the same number of uL of DNA to PCR #2, this higher template DNA concentration would make it more likely for there to be non-specific primer annealing to the template and could explain why you are now seeing these extra/non-specific bands."
I agree with Holie Rowlands answer....plz. standardise the dilution of 1st PCR product you can try up to 1:1000 dilutions, I read v old papers disccussing these small things but relevant .
I am attaching the paper..if you wish you can refer
Thank you, Hollie!
I put 2µl each 1st PCR products for being templates in the 2nd PCR.
My teacher gave me primers, so I did not check the annealing possibitlity. And even if it happens, I think my results from different times should be the same.
Thank you so much, Pratibha! I think I should give this suggestion to my teacher and let her decide and make another exercise for me.
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