screening of recombinants - (Sep/25/2007 )

hi
i have cloned gene with PEt vector with kan resistance
have got many(more than 200 for a plate ) transformants
in trhat i am feeling very difficult to scrren with colony pcr for all
if take directly pellet from colony to colony PCR i am getting no result
for that first i am isolating plasmid from all and going for pcr
it is becoming very difficult for isolation of plasmid and going again to pcr
please suggest any other method for screenig of recombinats among transformants
any other kind of colony pcr protocol directly from pellet for scrrening
plaese suggest
thanks in advance

It not clear how you perfom colony PCR. The widely used procudure is to pick colony (or take pellet from colony) into ~30 ul dH2O and boil for 5 min, centrifuge for 2 min and use 5 to 10ul of supernatent as template for PCR.

Hi Janu,

There should be no need to screen so many colonies for a routine cloning like this. You say you have 200 colonies on your ligation. This may or may not be good. The important this is whether you obtained significantly less colonies on from the control ligation (the ligation reaction without insert added).

From the control reaction, transformants can only be obtained from any undigested or self-ligated vector. This tells you how many background colonies to expect in your ligation. If the ligation plate has more than this background number of colonies, you can expect these to contain your insert.

e.g1 If the control plates gives 50 colonies and ligation plate gives 200 colonies
Around 50 of the clones on the ligation plate are uncut or self-ligated vector and 150 contain the insert.

e.g2 If the control plate gives 200 and the ligation gives 200
None of the clones in the ligation plates clones are likely to have insert - they are all background.

The ratio of colonies on the control and ligation plates tells you how many clones you have to screen. in example 1, 3/4 of clones are expected to have the insert so if you screen 8 clones you should get 6 positives (theoretically).

I don't recommend colony PCR - in my experience it creates more problems than it solves. I would go for the traditional alkaline lysis followed by digest to find the positive clones.