I am trying to screen a large no. of clones using colony PCR. I am using the standard and Tm optimized (for my gene) PCR protocol , PHUSION enzyme, other than longer initial denaturation. In all the reactions i am getting a smear in the lane and a band corresponding to my gene, if there, is very fade and hence difficult to observe. Can anyone suggest a remedy please.
Dear Amandeep,
Based on my experience, you get a smear in the lane if you use too much template (i.e., too much bacteria from the colony) in your colony PCR. If this is the case, you'll get very little or no product as well so this seems to match what you describe. Colony PCR only needs tiny tiny amount of material to work (you should barely touch the colony to take a sample) so my suggestion is for you to try to reduce as much as possible the amount of bacteria you use.This should improve the quality of your amplification and the smear should disappear. Hope this help!
Edwige
Dear Amandeep,
Based on my experience, you get a smear in the lane if you use too much template (i.e., too much bacteria from the colony) in your colony PCR. If this is the case, you'll get very little or no product as well so this seems to match what you describe. Colony PCR only needs tiny tiny amount of material to work (you should barely touch the colony to take a sample) so my suggestion is for you to try to reduce as much as possible the amount of bacteria you use.This should improve the quality of your amplification and the smear should disappear. Hope this help!
Edwige
I agree with Edwige. In addition, for a screening, you do not need an enzyme like the phusion. Use basic enzyme. In my point of view, the screening works well. If you screen for a gene in a plasmid, you could use generic primer such as SP6, T7 or pcDNA3, pGL3 etc... Often, the plasmids have this type of primer binding sequence. I think it is more efficient : you have a band at a low molecular weight for the negative colonny and a band at an higher weight for the positive colonny. No band means no amplification of the plasmid.
I agree with both answers. I faced the sample problem and reducing the template concentration solved the problem.
I would never recommend Phusion or any other enzyme in that class (PFU UltraII, etc) for screening clones. These enzymes are not very robust to differences in ions, amount of template, etc and they are expensive. Our lab uses KAPA2G Robust HotStart ReadyMix for colony PCR and it is amazing. You can have a little bit of cells in the reaction or a lot and it doesn't matter. I can't remember the last time I had to redo a colony screen.
I'm agree with the colleagues above. You can try disolving the colony in 50ul of deionized sterile water and then add 2 - 5 ul to each reaction, and you should use a regular DNA polymerase like GoTaq or similar.
Good luck!
Dear Amandeep,
I agree with Edwige, you can reduce you template. Dont use the same amount of template that you use in actual PCR for your genes. If still you have smears, you can dilute your template. If you have already screaned these methods, then sometimes smears appears due to high level of MgCl2 that you use in the buffer system.
@Edwige Moyroud... Thank you so much for sharing your experience, i will try it again today..
@Stéphane Roche.. Interesting thought..thanks
Sounds like we've all had the same basic experience. I agree with the earlier answers. We routinely use ordinary Taq for colony PCR. It's much cheaper than Phusion and works more robustly for that assay. Also, after heating the samples to release DNA we sediment very hard (14,000 xg/ 10 min) to ensure we don't pick up any particulates when we sample the supernatant for the PCR reaction. Carried over gunk can cause smearing. Also, if you use too much colony you'll be putting a lot of genomic DNA into the reaction, and that may also cause smearing.
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