Host cells for pBELOBAC11 - (Mar/02/2006 )

Does anyone know what E.coli strain should be used when using BAC-vector pBELOBAC11? I´ve used DH5alpha electrocompetent cells, but got no colonies.

Who has used this vector for cloning large fragments before?

Thanx for the help.

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Try DH10B strain. This strain is useful for BAC vectors.

Electroporate with a little bit higher voltage(15V per mm).

But in my case, DH5a was possible to harbor a BAC vector without insert, though it was not as efficient as DH10B.

Does your BAC have insert DNA? The larger the BAC is, less colonies you'll get.

Use larger quantity of BAC DNA for electroporation.

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Hi, yja97.

I´ll try DH10B soon. Thank you.

What do you mean with 15V per mm? I have used 2000 to 2500 V so far. The electroporator is an Equibio EasyjecT, perhaps you know this model. I´ve got no instructions on how to use it, so I just used an pre-installed program. When I did a positive control with just the uncut vector I received some transformants.

Thank you for your answer.

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I don't know Equibio Easyjet T. I usally use electroporator from BioRad. (EC 3 program )

mm means the width of electroporation cuvette. Usaully it's 2mm but there are another widths too.

15V was my mis-calculating. sorry. 3kV was treated to 2mm cuvette --> 15kV per cm, 1.5kV/mm

So, you'd better use higher voltage(3000V, if possible) when you're using 2mm cuvette.

But be careful. Higher voltage causes more errrors. Desalt your DNA completely, and make your com cells clean.

Good luck!!

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Thank you. Next time I´ll make a "gradient" with increasing power.

FMM

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