Hi!
I've been having problems transforming clones I made using the pSATN BiFC Gateway vectors generated by Tzvi Tzfira's group (doi:10.1016/j.jmb.2006.08.017), which I purchased from the Arabidopsis Stock Center into the Agrobacterium strain GV3101 by electroporation. I am wondering if anyone else who has used these vectors has faced similar problems and if so can you share with me how you overcame them.
My comp cells are fine as control vectors are easily transformed and yield the appropriate amount of colonies. I typically electroporate the cells and let them recover in LB (no antibiotics) for two hours, shaking at 28 degrees C and then plate on LB plates supplemented with Carb50, Gent30, and Rif25 and incubate at 28 degrees C. I have also just tried plating on Carb50 LB plates. After three days I get maybe 3-5 colonies.
Any tips, tricks, info anyone can give me on these vectors will be most appreciated!
Thanks!!
Hi Stacy,
In which binary plasmid did you subclone the expression cassettes from pSAT? Typically, the expression cassette from pSAT is transferred to a PZP-RCS binary plasmid, which is used to transform Agrobacterium with a Spec selection.
The whole pSAT system is explained here: 10.1007/s11103-005-0340-5.
Best regards,
Ahh - I thought they were already binary plasmids! Like pEarley. Thank you for the additional reference. I guess I have more cloning to do.
Hi, which entry vector did you use, once we do the BP reaction using gateway cloning, it is recommended to do the digestion of entry vector obtained from E.coli cells before proceeding to LR reaction, do the digestion and purify the product, then use that for LR. You should be fine in LR reaction and get colonies of E.coli, extract the plasmid and confirm the plasmid size on gel and/or by digestion, then use this plasmid for electroporation. you should be fine.
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