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Agrobacterium transformation not working!

Agrobacterium GV3101 Transformation pUC-SPYNE pUC-SPYCE

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#1 Erik Solhaug

Erik Solhaug

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Posted 03 August 2015 - 09:54 AM

Hi all-

 

I am trying to transform competent Agro cells (strain GV3101) with plasmids in order to do BiFC on them. I am using pUC-SPYCE(G) and pUC-SPYNE(G), which both are gateway-compatible versions of the SPYNE and SPYCE vectors generated by Walter and others (2004). Both of the vectors have ampicillin selection, but I have been using carbenicillin because apparently it is more stable. 

 

I am doing a freeze-thaw protocol where I put 1 µg of plasmid into 100µL of competent cells, then freeze the cells in liquid nitrogen and then thaw them at 37ºC for 4-5 minutes, then I put 900 µL of LB broth in the cells and let them recover growth at 28ºC for 4 hours, spin down the cells and make them up in ~200µL of fresh LB before plating 50 µL of cells on selection media.

 

I have plates with 1000X diluted Gentamycin (WC=50 mg/ml), Rifampicin (WC=50 mg/ml), and Carbenicillin (WC=100 mg/ml) that I have been using.

 

 

 

I did a transformation on Friday (7/31) using two sets of comp cells (ones that I had made in early July, and ones that were made by another lab) with a negative control (just added water), a positive control (added plasmid that I have been able to transform into Agro before) and a plasmid that I have been trying to get into Agro. So just to clarify, 2 sets of cells (mine, others) x 3 treatments (positive, negative, test) = 6 different plates.

I plated out the cells, and came back on Monday (after three days of growth) and found two things:

 

1) there was very little growth on any of the plates. Only a few small colonies were visible.

-A note on this point, I did use a Working Concentration of Carb that was double the amount that was suggested (WC of Carb is supposed to be 50 mg/ml), so this may help to explain the slow growth on all the plates.

 

2) however, even though the growth was slow, I could see small colonies on all of my plates (including the negative controls).

 

So I'm not quite sure what to do with this, please help!

 

Thanks,

 

Erik 







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