how to amplify Red/ET and Cre expression plasmid

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wildaisy's picture
how to amplify Red/ET and Cre expression plasmid

I am working with a BAC kit and want to amplify the Red/ET and Cre expression plasmid. These two are temp sensitive and will be lost at 37C. I tried to transform them into DH5-alpha, heat shock at 42C, then incubate at 30C for 1 hour, plate onto LB with Tc, and incubate at 30C for two nights. Cre plate did not grow. Red/ET plate had a few big colonies. I inoculated 3 colonies and miniprep DNA, but got very low concentration. Anyone has similar experience? Should I incubate them at 30C longer after heat shock? Why the conc. is so low?Anyone has the sequence and map information about these two plasmid? Thanks. pls reply here or e-mail to eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%68%7a%73%31%30%38%40%67%6d%61%69%6c%2e%63%6f%6d%22%3e%68%7a%73%31%30%38%40%67%6d%61%69%6c%2e%63%6f%6d%3c%2f%61%3e%27%29%3b'))

Fraser Moss
Fraser Moss's picture
The maps and sequences are

The maps and sequences are freely available at the Gene Bridges web site
Here is the link to the manual for the Quick & Easy BAC Modification Kit
You should download the specific manual for the product that you have here