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
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2 replies to this topic
#2
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Posted 18 April 2011 - 04:42 AM
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.
#3
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Posted 18 April 2011 - 11:12 AM
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
pSIM5 works definitly in BL21(DE3) ...thats a fact!
Good luck!
Regards,
p
