double digestion of transformed bacteria colony - (Nov/14/2007 )
Hi
I'm just wondering if anyone knows if double digesting straight from a picked colony differs from a double digest after miniprep?
thanks!
How would you do that? Ressuspending the colony in the mix and digest it? And then load the thing on a gel?
I don't think it will work... To cut DNA you have to extract it first, otherwise I don't believe the cells will incorporate your enzymes- unless you lyse them first. But even if you do, you'll have all the bacterial stuff in your mix and it will for sure interfere with your reaction. Moreover, even if the enzymes worked, they would also cut the genomic DNA and you'd have loads of bands in your gel. This of course if you saw anything on the gel - I don't think running lysed bacteria on an agarose gel is a very good idea...
I say miniprep it.
You wouldn't have enough of the plasmid to see on the gel. Minipreps not only remove all the cell debris (including the genomic DNA that your proposed reaction would also contain) but growing the culture overnight produces significantly more of your plasmid. You would need to grow up the DNA any way to have enough for good sequencing results and other downstream processes.
I'm just wondering if anyone knows if double digesting straight from a picked colony differs from a double digest after miniprep?
thanks!
Direct cell lysis and RE digestion of single colony is possible. But it requires specific reagents. Invitrogen has such product, clonechecker.
Hi everyone - thanks for your replies.
I was thinking of using a small part of a colony to confirm the presence of my insert while the rest of the colony will be used for LB overnight culture (reason: confirm insert right there and then without the need of waiting overnight for LB culture and also save miniprep reactions)
I did try picking a small section of a colony and innoculating it in my restriction digest mix. I did get slight digestion but most of the plasmid was still stuck at the well.
So i concluded that restriction digest straight from a colony is possible (but not very feasible)
I was thinking of using a small part of a colony to confirm the presence of my insert while the rest of the colony will be used for LB overnight culture (reason: confirm insert right there and then without the need of waiting overnight for LB culture and also save miniprep reactions)
I did try picking a small section of a colony and innoculating it in my restriction digest mix. I did get slight digestion but most of the plasmid was still stuck at the well.
So i concluded that restriction digest straight from a colony is possible (but not very feasible)
Why don't you try a colony PCR to check? That's what I do when I don't want to grow it up overnight, miniprep, digest. You can just touch the colony with a sterile tip into a PCR tube, add reagents, and just add a 10 minute 95 degree 1st step to lyse. Just make sure your primers work first.
I was thinking of using a small part of a colony to confirm the presence of my insert while the rest of the colony will be used for LB overnight culture (reason: confirm insert right there and then without the need of waiting overnight for LB culture and also save miniprep reactions)
I did try picking a small section of a colony and innoculating it in my restriction digest mix. I did get slight digestion but most of the plasmid was still stuck at the well.
So i concluded that restriction digest straight from a colony is possible (but not very feasible)
If your insert is big enough that you think you would see a significant difference in the size of uncut plasmid, then you can try to do a cracking experiment. Basically, you smear a colony into a buffer containing NaOH, SDS and sucrose, heat it and then run it on a gel. This will only work if your insert is big enough, and it won't tell you anything about orientation, just if the insert is there or not. f you want the whole procedure, let me know.
I second the colony PCR approach. This is such a time (and money) saver, especially when you have a large number of colonies to screen. It's best to use one primer specific to the vector and one for the insert: this way you can even screen for correct orientation in the case of a blunt ligation.
Ginger