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Transformant RFP expression

RFP DH5a Transformation

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#1 tihong10

tihong10

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Posted 25 February 2013 - 10:38 PM

Hello All,

I was performing a tranformation of DH5a with what I thought was a legitimate recombinant plasmid. The vector* i.e. plasmid without the insert has an AMP marker and a lacZ-red fluorescent protein fusion with a MCS in between the lacZ and RFP. After adding my ligation mixture with the chemically competent DH5a I plated on carbenicillin plates. Upon confirmation, I observed no growth on the control (non-transformed DH5a) plates and found two types of growth on the experimental plates. One colony type was cream colored and the other was red. I initially thought the cream colored colonies contained the recombinant plasmid because I was under the impression that an insertion in the multiple cloning site will severe the fusion between the lac promoter and RFP. However, I found out later that my primers were constructed incorrectly so that one of the enzyme sites was unrecognizable by the restriction enzyme.

Thus I am left wondering why clones grew at all on the antibiotic plates. If my insert and plasmid were double digested but didn't correctly cut one of the two restriction sites then how did colonies grow on the plate? Also, how does one explain the appearance of cream and red color colonies?

Also, am I right to assume that an insertion into the multiple cloning site of the original vector will result in no RFP production when the lac promoter is active?

If anyone can provide any insight into these questions I'd highly appreciate it.

THanks!




*Map of Plasmid Vector
http://www.snapgene....DsRed-Express2/





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