pSIM in BL21(DE3) - (Apr/18/2011 )

Hi guys

Is there any one who has tried pSIM5 in BL21(DE3)

I had been using pSIM5 in MG1655, W3110. I had little tough time with BL21. But then it works even with BL21

But I could not even transform pSIM5 to BL21(DE3). I ensured that I keep the plates at 30 and even I tried transforming the same plasmid parallely to MG and BL21(DE3). But everytime I fail to get colonies in BL21(DE3) even after 24 hours.

Can you guys help me please


vinu

Transformation efficiency of BL21(DE3) is very low -- about 2-3 orders of magnitude worse than cloning strains. How are you trying to transform it? Best would be to prepare plasmid from a cloning strain and transform with large amounts of pure DNA.

the competency of the cells in indeed a problem ...if you make them on your own check the competency by doing control experiments with e.g. pUC19. Use a defined amout of DNA and calculate the cfu's you get per µg plasmid DNA.

pSIM5 works definitly in BL21(DE3) ...thats a fact!

Good luck!

Regards,
p