Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Plasmid prep doesn't work! - Help needed with HB101 strain plasmid prep (Apr/21/2002 )

Hi,

I plan on screening a yeast genomic library (YCP50) but have been facing a lot of problems after amplifying the library. My problem is that i can't seem to isolate sufficient amounts of the plasmid for transformation into S. cerevisiae. I've tried the Promega Maxi Prep kit and the Qiagen Mini Prep kit to no avail. I would like to add that the E. coli is an endA+ strain (ie. HB101).

ANY help on this (frustrating) matter  will be greatly appreciated.

Thanks!

-y26404986-

Sounds like you are having fun.  Does the HB101 strain develp small colonies on plates ? Do you get any product at all from the kits ?
I have used the Wizard prep kits which give good yields, but I cant remember who makes them (I'll try and find out and get back to you).
You may need to collect more cells before you start. Take 1.5ml cells and concentrate them down by doing consecutive 30sec spins in 2ml eppendorf tubes until you have done maybe 4.5 -6 ml of cells in total.
Otherwise, you can go to the tried and true alkaline lysis.
Good luck.

-BJSAu-

Promega makes the Wizard plasmid isolation kit

-rcosgood-

You must check the replicon (the bit of DNA that is responsible for the number of times the plasmid replicates within the bug) of your construct . There are low copy number replicons and there are high copy replicons. If you are working with a low copy replicon the protocols you are working with offer solutions eg chloramphenicol amplification.  You should also probably switch to an endA- strain like XL-1 Blue.
Attached File

-milanoj-