Screening recombinant colonies - (May/19/2007 )

I try to clone 3 phytase genes: A, AI and B into E.Coli JM109. I use 2 vectors: pGEM-T and pPIC9K.
I plate the transformed cells on ampicillin dishes (100 ug/ml LB)
I have many colonies on the dishes

I use the following method to screen recombinant colonies:

80 ul cell culture (colony picked from petri dish and cultured overnight)
80 ul phenol - chloroform - isoamyl alcohol
33 ul loading buffer 6X
7 ul deionized water

Vortex vigorously

Centrifuge 10000 rpm, 1 minute

Use 20 ul of the supernatant to run on 0.8 % agarose gel

The control band (colony with only the vector) appears on the gel
However, I never get the recombinant clone ( although I repeat the experiments 5-6 times)

I wonder why colonies can grow on antibiotic dishes if they do not have the vector nor the vector+insert ?

The antibiotic is OK and there are no contamination in JM109 cells

Should I use another screening method ? (PCR colony or blue/white colony)

My English is a bit confusing, I guess. I am very sorry for that.

Do you mean that

1. you get a band for a colony which you have intentionally used as a control (transformed with the pGEM-T or pPIC9K plasmids), but not for any of the colonies which are supposed to contain your insert.

or

2. You get bands corresponding in length to the pGEM-T or pPIC9K plasmids for all your samples, but they are of the wrong length and contain no insert?

I assume you have turbulent growth in your overnight cultures. If they are not growing, then this is the problem.

I also assume you are growing the overnight cultures in ampicillin containing medium. If not, then the plasmid may be lost without selection.

I would suggest that the next step would be a normal miniprep of one of the cultures exhibiting this problem. I don't see any reason to think that the method you have outlined (which is a bit unusual) would not work, but when problems come up, fall back on the simple, reliable techniques. Carefully observe the size of the bacterial pellet you get from the control and your experimental colonies. You may be having severe growth problems due to toxicity.

Why do you say that this method is a bit unusual ?

This is the method my supervisor gave me.
She has sticked to this method for 2 months and there are no results !
She says that there's some problem with ligase. But I think there's some problem with the screening method.
Maybe there are not enough recombinant plasmids to give a band on the gel

Why not use a PCR screen and blue/white selection, as you mentioned? Not sure about pPIC9K but pGEM-T allows for blue/white selection. And a PCR screen is surely much easier than your current method! No overnight culture, for one thing.

Do you have the primers for the inserts? You can also do a screen by PCR, can't you? Just pick colonies and resuspend them in H20 (50ul each), then take 10ul as template and do PCR to see if you have the inserts?