Plasmid Editing

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mehdi71000
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Plasmid Editing

Post by mehdi71000 » Wed Jun 27, 2007 12:05 am

Hi I have a plasmid with two sali silts located on eater side of the desired gene. how can I cut out the other side so I’m left with the only the desired gene. I assume restriction sali enzyme is used. now how do I re connect them with another plasmid containing one sali site for the desired regulation I assume ill cut the plasmid on the sali site but how to join the two? :shock:
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canalon
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Post by canalon » Wed Jun 27, 2007 12:55 am

Hopefully there are closer restriction site in your plasmid B (in the correct direction depending on your regulator to insert the fragment of A that you'll have cut with Sal I.

Then you just need to add ligase, the right proportion of purified A fragment and B vector, the ligation buffer. Incubate a few hours at 14°C and you are ready to transform by heat shock or electroporation, then 1h hour in SOC at 37°C to recover and you just plate. Of course you have the right Petri dishes with the correct selective antibiotic marker, an incubator to grow them, and access to a way to get rid of biohazard waste...
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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MichaelXY
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Post by MichaelXY » Wed Jun 27, 2007 1:40 am

Wow, that stuff has me baffled. Just curious, what course do you learn things like that?

It sounds pretty advanced.

mehdi71000
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Post by mehdi71000 » Wed Jun 27, 2007 10:22 am

Thanks Patrick. You’re amazing. Love your stuff

MichaelXY wrote:Wow, that stuff has me baffled. Just curious, what course do you learn things like that?

It sounds pretty advanced.


I think it falls under genetic engineering category

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