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Transformed bacteria grows on plates but not in broth

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#1 goingtothesun



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Posted 14 September 2012 - 07:25 AM

I am trying to transform empty pLenti6.3/V5-DEST vectors in One Shot ccdB Survival 2 T1R competent cells and then grow up colonies for minipreps.

Here is a link to the vector information: http://products.invi.../product/V53306
And the competent cells: http://products.invi.../product/A10460

If you are not familiar with Gateway vectors, the empty plasmids contain the ccdB gene and must be transformed using the competent cells mentioned above.

Thus far I have been able to get colonies to grow on 100ug/ml ampicillin plates and 100ug/ml carbenicillin plates after doing the transformation. I cannot, however, get picked colonies to grow in LB broth with antibiotic.

Here is what I have tried so far.

1. Transformation, plated on 100ug/ml amp plates, tried to grow up in LB+100ug/ml amp but got no growth.

2. Made fresh amp plates. Made sure to cool to 55C before adding antibiotic. Used the next day for my transformation and got colonies. I had read a bit online and saw someone say they had to use 2x LB with the pLenti6.3 vector so I made some and tried the following. Each condition had 10 culture tubes:
LB + 100 ug/ml amp
LB + 100 ug/ml carbenicillin
2x LB + 100 ug/ml amp
2x LB + 100 ug/ml carbenicillin

Nothing grew.

3. Made fresh carbenicillin plates. Transformed, got colonies, and tried the following conditions:
LB + 100 ug/ml carbenicillin
2x LB + 100 ug/ml carbenicillin
LB + no antibiotic
2x LB + no antibiotic

I only got growth in the tubes with no antibiotic.

I am going to buy new antibiotics and make fresh plates. Does anyone have any other ideas that I could try?

#2 Curtis


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Posted 14 September 2012 - 06:04 PM

are you sure your stock antibiotic is fresh? ampicilin degrades fast, we prepare fresh plates every month. This used to happen to us a lot. we got colonies on plate but they didn't grow in broth. it was because false colonies grew on plate and when we transferred to broth with fresh antibiotic they couldn't grow.

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