Single Colony Gels? - (Sep/29/2006 )
Found this thread in the Molecular cloning forum. Cant seem to reach "zingmatter" but can anyone provide a protocol for this single colony gel?
It would link nicely into a previous diccussion: http://www.protocol-online.org/forums/inde...showtopic=20511
21 2005, 10:54 AM
As blunt-end ligations are quite inefficient, dephosphorylation will make it even more inefficient.
Personnally, I won't de-phos at all. Yes you'll get squillions of ligation positives but you can screen 50 - 100 very easily using single-colony gels (or blue/white selection even better of course if you have it). A single colony gel (if unfamiliar) is simply a blob of +ve colony lysed in SDS buffer (with a dye), spun and ran on a large agarose gel. An insert will cause the supercoiled plasmid to run slower than plasmid w/o insert. I've used this technique to detect a 50bp insert in a 6kb plasmid. zing
Colony PCR is certain an interesting idea... a pre filter before colony PCR. I have not the chance to try this protocol, as my ligation of late have been rather small insertion (300bp). I would be interested if somebody had the time and to try this protocol out. (It would be another week before I get colonies to try this protocol out)
From Zingmatter's comment, I have speculated that the lysis buffer is simply, the standard lysis buffer for E coli, 0.2M NaOH and 1% SDS.
My feelings would be that half the colony would go into the lysis buffer. A little mixing, and the entire contents run on a 1% agarose gel. I am however uncertain what the 0.2M NaOH would do to the gel. The salt content is a little higher then anything I have tried before.
Perhaps grow each colony in 1-2ml of LB broth for several hours. Use half of this to run on the gel and the other half can then be made up as a glycerol stock in case it works (fingers crossed). Would it be as simple as lysing the E coli cell, centrifuging the debris and running the supernatent?? Also would the different sized plasimd even enter the gel to run through the gel?? What if the 0.2M NaOH was neutralised before running?
I do see the point of not having sufficient DNA to visualise the DNA bands. However I am not to keen on investing time in a futher growth phase. 14hr of waiting for the cells to grow up is enough as it is. I propose that the problem of insufficent plasmid DNA be solved by running the lysed cells in a very narrow well .
Jaff, there is not need to make glycerol stocks, the e coli cell will easily survive 2 days in sd water and easly a month in LB (at 4 Celsius)
Ideally, I won't even centrifuge the cell debris. Simply lyse cells and run supernatant. The gel should catch the protein and denatured genomic DNA.
Yes, a valid point, ideal conditions aside... would the plamid DNA be able to pass through the protein and genomic DNA junk, and enter the gel? .... I am not sure.
Finally, I don't think neutralisation of NaOH would help. It is still salt and whould interfere with gel running. Or taken it another way, i also feel it is unlikely that there are sufficient ions in the E coli cell to neutralise that quantity of NaOH.
I've tried a similar protocol once, a single colony gave enough DNA to visualise on a gel, but as I needed the right direction which I could detect with colony PCR, I later only performed the latter.
If I remember correctly (will look it up tomorrow), you lyse the e. coli in a pretty standard lysis buffer, but with addition of bromo-phenol blue and sugar (as you need a loading dye, you get it in your lysis buffer at once). Just resuspend the colony in there (don't forget to strike on a fresh plate or inoculate small amount of LB), incubate for some minutes, centrifuge to pellet debris, load supernatans on gel.
make a lysis buffer with 10% Sucrose (W/V) and 100 mM NaOH, 60 mM KCl, 5 mM EDTA, 0,25% SDS and 0,05% Bromophenol blue
Prewarm the buffer to 40-45 °C
pick colony and streak on a master plate
transfer the remainder of the colony to 30 µl prewarmed lysis buffer
incubate @ 40-45°C for 5 minutes
place on ice for 5 minutes (to precipitates K and SDS)
spin 1 minute @ max speed in a microcentrifuge
load on gel (15-30 µl)
Has worked for me, though colleagues have told me that they also had a method including the use of lysozym that has worked as well.
Thanks for the protocol, I think it worked. Got a lot of junk DNA which either stayed in the wells or ran out with the dye front. Got nice bands at the approx. size I was expecting. The samples themselves entered the gel and ran in a smoothly in a straight line
Thanks for the protocol, I will try it today!