I am facing problems of non specific amplification in my colony PCR experiments. My specific product is present but its band is not as intense compared to the non specific mesh that appears below it. My product size is 300bp, whereas the mesh appears near 100bp and is very intense.
These are the steps regarding my experimental protocol:
1) Transformation of ligation mix in DH5alpha
2) Pick 5 transformant colonies
3) Grow each colony in respective antibiotic containing LB broth for 5-6 hours
4) Took 500ul of culture and centrifuge
5) Resuspend in 50ul of nuclease free water.
6) Denaturation at 95 C for 10 min
7) centrifugation of lysate at high speed for 5 min.
8) Use supernatant as template for colony PCR.
9) PCR reaction:
go green master mix = 10ul
gene specific primers (10uM)= 2ul each
template( supernatant)= 6ul each
Note: I am getting good results by following same protocol for other constructs.
If you are getting non-specific amplification, try a gradient PCR and also a touch-down PCR. If you get a band in the negative control, then it is contamination from the mix. How does the gel look especially the one between the positive and the negative control?
If you are getting non-specific amplification, try a gradient PCR and also a touch-down PCR. If you get a band in the negative control, then it is contamination from the mix. How does the gel look especially the one between the positive and the negative control?
Positive control reveal discreet band with no smearing where as negative control has no bands. Thats why it is very confusing for me. :(
Agree with Lekha, you need to improve your PCR experiment conditions (temperature actually). For your positive control you use purified template (right?) and for colonies screening - total DNA extract (with bacterial genomic DNA), so your primers can be complementary to another template at used temperature. Try gradient or new primers.
Good luck!
Test your primers with purified template in a gradient PCR to obtain the optimal annealing temperature. Also, diluting the template 10 fold in water can often improve PCR results. I take it you are centrifuging down 500uL of cells and re-suspending in 50uL of water before denaturing, spinning and using the supernatant for PCR. You are using a lot of cells and there is probably a huge carryover of salts. This will affect the annealing temperature of your primers and give different results to your control which is probably purified DNA. I typically just dip the end of a pipette tip into my overnight bacterial culture and swirl it in the PCR mix. This generally works well as you only need a few cells for the PCR.
Pick colony, streak in PCR well, and then streak on plate as backup. Add master mix and do PCR. There is no need to grow. After PCR, grow up positive colonies ON for prep. Best is to perform colony PCR using a vector primer and insert primer. If you use insert primers only, you can amplify ligation mix that is all over the plate. I have had this several times and now avoid doing that.
Maybe it will help to run a BLAST with your primers, because you might be amplifying something on the genomic DNA. If that's the case, you should change your primers to be as specific as you can to your plasmid only. I also agree with Koen Venken, the best thing to do is use one primer on the vector and the other one on your sequence of interest. I also run my colony PCRs directly from the plate and it works great.
Check the mix without DNA or primers. I am agree with Koen Venken and the PCR could be made with excellents results using in a mastemix PCR and put the colony inside the reaction.
Dear Siddiqui,
It seems to be a PCR optimization problem. Just standardize your PCR conditions specially annealing temperature and use touch down PCR if needed.
best of luck
There are two reasons generally: (1) the PCR condition, especially the Temperatures of anneal, overhigh and too low temperature of anneal all cause unspecific amplification. So the gradient PCR (set different anneal temperatures) will help you.
(2) The primers. if the primer are not only bind the special site, it will cause unspecific amplification. So when you design primer, the wise method is that design two or three pairs primers.
Hi
I think colony lysis is most important parameter in colony PCR. I face similar problems. What we can do is just take fresh colony (one day old max.) and suspend it in sterile dist. water. Add Tween-20 (0.05%), mix and boil for 10 min. Centrifuge and take 2 ul for 20ul reaction. I get band, you will too. And yes, routine Taq is good enough. Good luck.
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