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restriction problem


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#1 bsengez

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Posted 13 December 2009 - 09:19 AM

Hi,
I cloned my genes into pGEX-2T expression vector. I checked my inserts via colony PCR and the results are okey. But when I digest the plasmid to see my insert, it becomes a problem. There is something at expected level, but it is not an exact band, something imaginary. I checked all my stocks, primers, transformed it (I used BL21 compotent cell, because I will use the vectors in protein purification. Does it affect restriction enzyme efficiency?) again, but all same. I cannot solve the problem. Any help would be appreciated.
Burcu

#2 pito

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Posted 13 December 2009 - 10:04 AM

can you upload a picture here from what you see?

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 phage434

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Posted 13 December 2009 - 10:59 AM

You should not clone directly into BL21 cells. The transformation efficiency is very low, making the transformation harder than it should be. Also, the quality of the plasmid DNA prepared from the cells is low, due to the presence of endA endonuclease. I don't know what an "imaginary" band looks like, but it doesn't sound like something I'd want to depend on.

#4 bsengez

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Posted 13 December 2009 - 01:56 PM

These were my constructs from previous years. I have made subclones, transformed them into DH5alpha and sequenced. Everything was okey. I just want to check my stocks in BL21 whether something is wrong, but I forgot that BL21-DE3 competents have endA, really thanks.


You should not clone directly into BL21 cells. The transformation efficiency is very low, making the transformation harder than it should be. Also, the quality of the plasmid DNA prepared from the cells is low, due to the presence of endA endonuclease. I don't know what an "imaginary" band looks like, but it doesn't sound like something I'd want to depend on.






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