Transformation problem (HELP!) - (Aug/24/2005 )

I tried to ligate double digested PCR product to pET23a(+) vector.

After double digestion, i ligated digested insert to digested pET23a(+) vector. then i transformed the ligated products to DH5a E.coli cells.

However i couldn't get any colonies from the plate.

I already checked the CP cell efficiency and the results showed me its working very well.

i tried this experiments about 5~6 times with different ratio and time.

After all, i could get 2 colonies. and then i sequenced it.
Fortunately it was the right sequence which i planned to insert to the vector.

However The real problem has stated,

I tried to transforme the vector to BL21(DE3) CP cell for protein expression.
But i couldn't get any colonies....

i check these already.
1. I transformed the inserted vector( after prep) to DH5a
---------> i got a lot of colonies
----------> with those colonies i get the right inserted vectors

2. I trasnformed the not-inserted commercial vector to BL21
----------> i got colonies



What do you guys think is my problem? T.,T

Help me

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First thought - toxic product. BL21 does have a very small amount of leaky expression, at least in my experience. If the expressed species is toxic, your cells will die before they get a chance to grow. Possible fix - try transforming into BL21(DE3) pLysS strain to reduce the leaky expression.

Second thought - plasmid size. If the insert is especially large, that can affect transformation efficiency. If the whole construct exceeds a certain critical size, the bacteria won't be able to take it in during the transformation. I doubt this is the problem though, as your insert would have to be gigantic.

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Transform again, and keep the plates! I had the same problem, so I kept the plates in the incubator for another night. The next day I had an ample amount of colonies. Later I cultured a batch of untransformed BL21 together with my transformed BL21 cells, and I noticed that the transformed BL21 cells grew much slower than than the native cells. I should also mention that I'm working with a pGEX plasmid that have very high leakage, so the cells continuously produce protein. Luckily my protein is not toxic, so the only adverse affect is that the cells grow slower.

And also, make sure to use a very rich medium, 2xYT works fine for me, both in cultures and in plates.

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Thank you very much..^^

AS you said, The problem was 'Slow growing of Transformed BL21DE3'

After i grew it for 2 over night, i could get many colonies, and After

i extract plasmid DNA, It was the right one i wanted to transform.

Now i'm trying to induct specific proteins from these cells..

^^

Thanks again and Have a nice day ~

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You can also add glucose (~50mM) to the media to help repress the gene better.

Daniel

Molecular biology troubleshooting

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