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Help with cloning a plasmid that contains repeats!


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#1 mchotalia

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Posted 04 March 2009 - 09:21 AM

Hello there,

I have been provided with a plate of bacteria containing a plasmid with multiple repeats, because the bacteria insist on recombining parts of the plasmid/or spitting it out all together i am culturing the colonies at 30oC rather than the typical 37oC. I have now attempted this three times and each time the restriction pattern does not look as expected i.e. the bacteria keep recombining the plasmid. Does anyone have experience with this? If so are there any tips?

All answers appreciated

#2 perneseblue

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Posted 04 March 2009 - 10:55 AM

Hello there,

I have been provided with a plate of bacteria containing a plasmid with multiple repeats, because the bacteria insist on recombining parts of the plasmid/or spitting it out all together i am culturing the colonies at 30oC rather than the typical 37oC. I have now attempted this three times and each time the restriction pattern does not look as expected i.e. the bacteria keep recombining the plasmid. Does anyone have experience with this? If so are there any tips?

All answers appreciated


What E.coli strain are you using? Have you tried changing E coli strain?

What kind of plasmid are you using to carry your repeat structure? Try using a single copy plasmid. (if you haven't already).

How are you growing your cells? Try using a bavelled flask. If unavailable, try growing your cells in a flask 10x the culture volume.

When are you talking off the cell culture? Try reducing growing time to 14hr.
May your PCR products be long, your protocols short and your boss on holiday

#3 scolix

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Posted 06 March 2009 - 11:09 AM

Try cells like SURE cells, Stbl3. These cells have low levels of recombinations. these can help.




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