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Creating empty vector by self-ligation


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31 replies to this topic

#31 chijing

chijing

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Posted 06 June 2009 - 06:53 AM

Hi,

Could you please share with us how you solved your problem?


Regards

GH



Hi GH,

hmm..basically..i did a double digestion on the pCMVSport-6-insert using the SalI and xhoI( these create compatible ends which can be ligated later). Then i ran it on 1% agarose gel and cut out the 4.4kb band (which is the pCMVsport-6 empty vector backbone). Followed by gel extraction and the purified plasmid DNA was then religated using T4 ligase, incubate for overnight at 16 degree celcius. Then i transformed 10ul of the ligation mixture into E coli DH5alpha. I managed to get colonies on LB+Amp plate.

That's all of it.

Best of luck.

Regards,
CHi-Jing

#32 kellyhayes

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Posted 23 November 2009 - 04:05 AM

There is a huge difference, the supercoiled form runs lower. Initially i used 6ug of plasmid DNA for double digestion and i assume i lost some during the gel extraction process.

regards,
kelly

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