Hello everyone, I am trying to overexpress a protein into BL21DE3 pLysS and have successfully cloned the gene in DH5α and checked the plasmid via double digestion to check the fallout. The concentration of the plasmid is 150ng/ul and I have diluted it two folds to reduce the concentration to half i.e 75 ng/ul. I am using 1.5ul of the diluted plasmid in 50ul of competent cells and plating on Cat-Kan LBA plates having 34ug/ml and 50ug/ml concentration of antibiotic. However, I am unable to see any colonies on the plates. kindly suggest what could be done to get the transformation done.
I would suggest you increased the amount of competent cells you are using (especially if they are made in house). Generally, Ecoli B derived strains are more difficult to transform than Ecoli K12 derivative (especially when the plasmid was prepared in a K12 background).
Hi,
What were the parameters used for the transformation? And plasmid size?
In the past I had problems in obtaining colonies because the E.coli lost the competence/lost the efficiency for transformation, and in the and I produced new competent cells.
I have tried transforming using 100 ul of competent cells as well as 50 ul of the same @Hanna
@Leandro Initially I diluted my plasmid two fold yielding a concentration of approx 75 ng/ul. I used 1.5 ul of the same and transformed into 50 ul competent cells. My negative control is competent cells without my vector. So technically in the negative control colonies shouldn't grow on Cat-Kan plate because of lack of kan resistant plasmid and I don't see a growth likewise. Initial incubation for 1 hr with plasmid added to comp cells followed by heat shock 90s and incubate on ice thereafter for 4 min. Add 1 ml LB and recover cells for 1 hr at 37 degrees incubation. Finally resuspend cells in 50ul LB and plate them.
You use a standard protocol and the sizes are normal, it should be working.
I suggest doing treatments to make them competent
try to decrease the antibiotic concentration and make a negative control and compare between the control and your experiment.
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