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Erythromycin LB agar

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#1 valen



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Posted 23 June 2011 - 05:07 AM

I am using pTRKH3 plasmid http://bccm.belspo.b...s.php?NM=pTRKH3 .
I am trying to clon a cDNA (fused to GFP) in pTRKH3 but when I do the transformation (using DH5alpha) with Erythromycin (150ug/ml or 250ug/ml) the plate is full of bacteria (there are no colonies).

Could you tell me what's going on?

Many thanks in advance,

#2 leelee



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Posted 23 June 2011 - 07:26 PM

Where in the parental pTRKH3 haveyou inserted the cDNA/GFP?

Assuming you've done so somewhere not disrupting the erythromicinR gene, this is what I think:
-Your transformation efficiency is high so you are getting a lawn of bacteria that have taken up the plasmid and are resistant.
-Either take a loopful from one of the lawn plates and streak out onto a fresh plate for single colonies. Or repeat the transformation and spread plate various amounts of the transformants (eg 100ul, 50ul, 20ul, 10ul per plate) so that you will end up with a plate with a low enough amount of bacteria for single colonies to appear.

Option 1- You are transforming bacteria with a ligation product, that may still contain circular pTRKH3. So the parental plasmid (pTRKH3) as well as your plasmid containing cDNA/GFP are erythromicin resistant. So after transformation you are seeing growth of bacteria containing either of these plasmids. You should look at using the tetR (if you've disrupted this with your insert) to find your cDNA/GFP transformants.

Option 2- you know that the insert cDNA/GFP has been correctly inserted (without disrupting erythromicinR) and there is no possible parental plasmid carryover/remaining in your DNA prep.

#3 phage434



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Posted 24 June 2011 - 05:37 AM

The strain you are transforming may be erythromycin resistant. Check this by plating untransformed cells on your plates. The plates might also be bad -- is the agar cooled prior to adding antibiotic?

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