Colony PCR - (Jun/19/2007 )
I have a question about colony PCR. I don't get any positive results.
Does anybody know what is the best protocol is for E.coli (BL21 + pET21) colony PCR?
At the moment, I prick a colony, put it in 25 ul of milliQ. I use 1 ul of this mix as PCR template.
Before the PCR, I inserted a step of 8 min heating (98 degrees).
I it necessary to use lysozyme or something?
After heating the sample, centrifuge to move the cell debris to the bottom of the tube and add 1 uL of supernatant to the PCR reaction.
I usually put my bacterial sample into 5µl Triton-X (1%) and after a short incubation I fill it up to a 25µl-reaction with the other PCR components...
i don't do anything.
I dilute my colony is 50ul sterile water and use 2.5ul of the solution and add that to 7.5ul PCR mix. I heat at 95 Celsius for 4min. There is no need to use lysozyme, the heat and the detergent in the PCR buffer is good enough to lyse the cells
It is important not to use colonies that are too old in colony PCR. Big large old colonies do not PCR. (Note: I don't spin down the cell debrie. Thus the importance of not using too much cells.)
Lastly reduce the heating. 98 celsius is far too high. The Taq enzyme will probably not be happy at such high temperature. How long is your PCR product?
i boil my colony at 95 celsius. spin down to get rid of cell debris.
i use the same bacteria to do PCR using boiled colony and genomic from phenol chloroform/kit extraction. sometimes you need extra steps to do colony PCR as there is more 'junk' compare to a proper extracted genomic using kit/phenol chloroform. using the same protocol, i can get result for extracted genomic PCR, but no band at all in the PCR using boiled colony. but after i put some PCR additives into my PCR mixture, boiled colony PCR gave me a nice band that i want. maybe you can try it: prepare a mixture of 10% glycerol, 10% formamyde and 10% DMSO. for eg, your final volume of PCR is 25ul, put in 2.5ul into your PCR mixture.
I also have a problem with colony PCRs I am running. I thought I would post it here instead of making a new thread.
I have tried this different ways, and both normally worked for me.
The most recent time, I made 100ul of 1Xphusion PCR mix. Put 10 ul into each tube, then picked a small amount of colony and put it into the respective mix. Made the initial denaturation step 95C for 5 min. I then ran all 10ul on a gel, and got smears instead of bands. Any advice?
The usual problem is far too many cells. I use a 10 ul tip and touch the end to a colony, then mix with 50 ul of DI water. I use the same tip to inoculate a gridded master plate. I add 0.5 ul of the water to a 10 ul PCR reaction, and lengthen the inital heating to 5 minutes at 95C. I usually run 36 cycles with a taq/pfu supermix (we use the Invitrogen PCR Supermix High Fidelity). It's important to optimize the PCR reaction ahead of time so that it is robust in the presence of a lot of contaminating genomic DNA, or to use standardized primers that reliably work.
Yes, too many cells (or old cells) is the main problem, but if you're careful you can swish your toothpick tip in the PCR mix directly, and skip the dilution step. I found this worked much better with 20ul reaction volume than 10ul, presumably because with 10ul the concentration of cell debris is too high.
Hmm can we make a pinned thread on Colony PCR? I kept on reading basically quite the same thing.
It seems alot of similar threads running around.... haha
v will follow that add prick of colony to 50ul of sterile dis water and heat it in boiling water bath for 10 min and keep it in ice for 2 min.. then centrifuge for 5 min in max speed at RT... then 2ul of this as template and remaning 8ul as PCR mix.. totally 10ul.. u get good conc of recombinants.. first transformation patching should be done to get isolate recombinants..