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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 







Also tagged with one or more of these keywords: Agrobacterium, GV3101, Transformation, pUC-SPYNE, pUC-SPYCE

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