how many colonies to screen? - (Mar/30/2007 )
A couple of questions:
Are there 'general rules' for how many colonies to screen before you decide that the ligation + transformation didn't yield the construct you're looking for?
I've got a plate with hundreds of colonies for my vector + insert, and my control plate (religation of de-phosphorylated double cut vector) contains just 4 or 5 colonies, but I don't seem to have the right construct. Say from a plate that contains 200 colonies, how many do you screen before you accept that your construct probably isn't there?
Also, I have some very peculiar results that I sometimes have seen after a ligation+transformation, and I don't know how to explain this:
After linearizing the plasmids from individual colonies, I run them out on a gel along with linearized vector, and uncut controls of each of the plasmid isolations. I cut the supposed vector+insert plasmids with the same enzymes that I used to open my vector/cut my insert. The gel lanes are as follows:
1 - uncut vector
2 - uncut plasmid #1
3 - double cut (EcoRI, HindIII) plasmid #1
4 - uncut plasmid #2
5 - double cut (EcoRI, HindIII) plasmid #2
6 - uncut plasmid #3
7 - double cut (EcoRI, HindIII) plasmid #3
8 - uncut plasmid #4
9 - double cut (EcoRI, HindIII) plasmid #4
10 - uncut plasmid #5
11 - double cut (EcoRI, HindIII) plasmid #5
I'm 100% sure I loaded the gel in that order, and this is the result:
1 - single band, 5900 bp
2, 4, 6, 8, 10 - Two bands one running above the uncut vector, one running below, both bands are a bit smeared (probably just too much DNA I guess)
3, 5, 7, 9, 11 - one band, about half the size of my empty vector.
At first I thought I had simply loaded the gel in the opposite alternating order than I had intended, but I really don't think I did.
Any ideas at all?
Intact plasmid has different conformations in agarose gel. It is not unusual to get more than 1 band for an intact plasmid.
May I know what is the size of the fragment you are trying to release?
It is not a good idea to do digest to all the extracted plasmids. First, it is a waste of enzymes. Enzymes are not cheap unless you come from a luxury group. Secondly, you are just wasting your time and energy.
I would suggest you to run your extracted plasmids along with your original intact plasmid. From there, select only those plasmids, which appear higher than the original intact plasmid, to set up restriction digest.
Regarding your question on how many colonies to screen. There is no specific number of colonies you need to screen in order to make your result valid. After all, you only need one construct. Normally, I would screen for about 12 colonies. If I am very desperate to get my construct, I may screen up to 24 colonies or more, depends on my feeling on that day.
Thanks for your input, the size of my insert is ~1300 bp
I would screen 200 colonies before giving up on that ligation/transformation. But I screen using colony PCR and my ligations are generally multiway ligations, thus efficiency is low 1:20 - 1:25. On principle I would screen no less then 24. My average screen size would be 72 by colony PCR
For really difficult ligations, my lab mates would screen 4000 colonies (by colony hybridisation) before giving up on the ligation mix. But their ligations are like ligating 20kb fragment to 20kb vector.
As you are screening by digest, I would suggest you curtail your use of enzymes. If possible use a single enzyme whose banding products is infomative, eg if your product is present it gives 3 bands. If absent it give only 1 band.
I would absolutely do a colony PCR rather than digestion screen. It's much less effort and far cheaper.
How are you guys doing your PCR screens? I'm not confident at all that a PCR screen isn't subject to a high rate of failure. Could you please post your protocol and reagents used?
I've tried it a couple of times and always the results aren't great (i.e. not as definitive as I would like the results to be, I always get hazy bands).