4 replies to this topic

[satishbty]

Hi ,I am using TSS solution(2X LB ,PEG,MgSO4,DMSO) and TCM (CaCl2,MgCl2,)for transforming DH5 Alfa cells.From overnight grown cells I am taking 1:200 dilution to get .15 to .2 OD600 . centrifugation 2500 rpm for 10 minute(room temperature--no access to 4 degree centrifuge ) I am using 1/10th volume of TSS to resuspend cells.keeping cells in TSS and ligation mix in TCM for 1 hour on ice separately(20 micro liter ligation mix and 180 TCM ). Mixing them and 1 hour incubation in ice,.heat shock 42 degree 45 sec.wait 5 min. SOC 600 microl.and plating on antibiotic plate. I am not getting single colony on plate.Any suggestion where I am missing...

[phage434]

Usually even bad transformation yields a few colonies.  Make sure your plasmid antibiotic resistance and the plate antibiotic agree.  Are you recovering the cells for an hour on non-selective medium? Can you transform other cells with this plasmid and the same plates?

[satishbty]

phage434, on 19 January 2011 - 05:57 AM, said:

Usually even bad transformation yields a few colonies.  Make sure your plasmid antibiotic resistance and the plate antibiotic agree.  Are you recovering the cells for an hour on non-selective medium? Can you transform other cells with this plasmid and the same plates?

    
yes .i am growing them on SOC for one hour.My ligations are ok.antibiotics are ok.

[phage434]

So, are you trying to transform ligation products, or purified plasmid?  If you are having trouble, I would highly suggest transforming serial dilutions of a known working plasmid, rather than starting with ligation products.  Too many things can go wrong with ligations, and you want to separate the source of the problem.  You should be able to easily transform 100 pg of purified plasmid DNA to get hundreds of colonies.

[satishbty]

phage434, on 19 January 2011 - 02:14 PM, said:

So, are you trying to transform ligation products, or purified plasmid?  If you are having trouble, I would highly suggest transforming serial dilutions of a known working plasmid, rather than starting with ligation products.  Too many things can go wrong with ligations, and you want to separate the source of the problem.  You should be able to easily transform 100 pg of purified plasmid DNA to get hundreds of colonies.

  

I will try with this .thanks for suggestion.