Hi everyone, I need some help with my colony PCR procedure. Last Week I did a heat shock transformation on E. coli DH5alpha with pUC57 plasmid. I extracted the DNA from the bacterias by heat, however, the colony PCR shows primers dimers. I don't know what happened 'cause i'm sure the primer design is correct for my gene. The primer design is for Gibson Assembly though, and I used a high fidelity DNA polymerase. Some advices?
PD: In the image, the first well it's the positive control.
How did you perform colony PCR? Did you use the whole colony in the PCR reaction mixture, or did you use a diluted fraction of the colony after dissolving it in sterilized nuclease-free water?
For colony PCR, we usually dissolve a colony in 10–20 microliters of water and then use its 1 microliter as a template for PCR. You can increase the initial denaturation time to 10 minutes so that DNA can be better accessed for PCR. If you're still not getting results, it's okay. You can isolate the plasmid and then perform PCR. Best wishes!
María Fernanda Rojas Hernández Hello there! What are your PCR conidtions?
Looks like bacterial cell lysis did not happen.
Two possibilities, the first is the PCR failed, the second is that none of your preps are correct. Given how clean the gel is though, I think most likely there is a PCR issue. Check that you really have template DNA and didn't lose it along the way. Be sure not to use too much template, double check your conditions and programming, and make sure your PCR reagents are good. If you run some appropriate controls you should be able to diagnose the issue.
From your positive control, it seems that your PCR reagents are good enough. Instead of your gene specific primers, try PCR with vector specific M13_forward20_primer & M13_reverse_primer pair. For colony PCR you need not do thorough DNA extractions. for high throughput quick colony PCRs, just touch individual colony with a sterile microtip (preferrably 10 uL tip) and then dip and swirl it in the corresponding PCR tube reaction mix. This always works with 25 uL PCR reactions for us. Do not forget to run a positive control with proper kit extracted plasmid (same as you are using here) with insert of known size (may be from previous experiments).
best wishes,
SD
When the bacterial colony is taken from the Petri dish, care should be taken not to poke into the agar, because the agar contains taq-polymerase inhibitors, and also diverse enzyme inhibitors...
We can also replace the agar with agarose, in the Luria Bertani medium, which could avoid this inhibition of PCR.
Gibb AP, Wong S. Inhibition of PCR by agar from bacteriological transport media. J Clin Microbiol. 1998 Jan;36(1):275-6. doi: 10.1128/JCM.36.1.275-276.1998. PMID: 9431965; PMCID: PMC124852.
Best wishes.
That positive control looks a bit odd, the band sizes are larger than your ladder and you are getting several bands. It looks similar to just plasmid. What are you using for your positive control template? If it is pure plasmid, be sure to dilute it a lot (like 1:100 of a standard miniprep, then use 1 ul as a template).
I would vote for reworking the positive control and trying again.
Hi all, I'm currently doing colony PCR as well but all of my bands get smearing, so for second time I only take a little (barely touch) colony, but there is no band, I'm think is it because I take colony from MacConkey agar that contains bile salt or inhibitor that affect PCR? first I take the colony and dilute in 15uL water and then the reaction mixture is 50uL includinhg master mix and primer, should substreaking the colony on nutrient agar first before I do PCR?
Also try to replace agar with agarose in making petri dishes ...
It's a little more expensive, but it's worth it ...
Good luck !
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