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jpngl's picture

Dear sir/Madam,
                            I have tried to amplify a A. tumefaciens LBA4404 harbouring a contruct using screening primer specific to that gene and i havnt get any amplification, but the same which was in E.coli, displayed a nice amplification using same pcr profile, master mix, colony concentration etc....what could be the reason for not getting amplification of that gene in LBA4404, is there any treatment over the colonies in A.tumefaciens strain? because, i read in a paper that treating with NaOH will increase the efficiency of amplification. If so, can anyone provide me the details for the treatment if neccessary?

varsha's picture
It is likely that the DNA was

It is likely that the DNA was not extracted from Agrobacterium colonies under the conditions of  PCR.
Try protocols in these papers:
Osman, F. and Rowhani, A. 2006. J. Virol. Methods 133: 130-136.

Jason King
Jason King's picture
Have you done colony PCR on A

Have you done colony PCR on A. tumefaciens before? When I've done it in the past with E.Coli, I have just used a pipette tip to transfer part of the colony to the PCR tube and then relied on the heat to crack open the bugs. Is it possible that A. tumefaciens are more heat resistant? 
Is the gene you are screening for an endogenous gene or is it present on a plamid that you have introduced? If it has been introduced, are you sure that the colonies are antibiotic resistant?

A Timmer
A Timmer's picture
Some of the bacteria we do

Some of the bacteria we do colony PCR are on are difficult because there are inhibitors as well. So we try to make sure we don't add too much of the bacteria to each reaction. We usually pick one colony into 10 ul of water and microwave this for 2 minutes. Then we add 0.5-1 ul of this to each PCR reaction.
Maybe you should try a positive control in the A. tumefaciens to be sure that your colony PCR protocol is working.

Shubhangi's picture
Colony PCR is a very easy and

Colony PCR is a very easy and reliable method to screen clones.  For E.coli I resupend a single colony in 20 microlit H2O. Vortex it and keep in boiling water for 2 min. Spin at 1000 rpm. take sup to set up a PCR. Spiining might be useful as you remove debris and larger proteins.
Including proper controls can sort where problem is. Is it with your bugs are more resistant or reagents are gone wroong or simply transformation has not worked properly.