Question on genetic cloning

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sofiash
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Question on genetic cloning

Post by sofiash » Mon Mar 13, 2006 7:13 pm

Hello everybody!

I have a question.After doing a transformation with some vector into an

animal cells, how can I establish how many times there was an

integration of the vector into the genome and the exact number of

sites where the integration happened.It is a generall question,without

knowing the specific vector and the type of the cells.

How can it be done and what kind of technics should I use in order to

receive the most acurate results?

Thanks

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damien james
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Post by damien james » Tue Mar 14, 2006 3:54 am

This is experiment that you are doing? If so, I am very interested in your parameters. From experience, I only know about bacterial DNA uptake, and is usually shoot and miss when describing genetic integration and induced mutation. An educated guess is made based on new biological characteristics from differential culturing. Knowing exact number of sites would be very hard because most of uptake is in non coding region. You could try radioactive or fluorescent markers attached to inserted DNA.
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canalon
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Post by canalon » Tue Mar 14, 2006 4:24 am

quantitative PCR might work to give an estimate of the number of integration with the right standard.
For exact location, I would say something like, restriction followed by pulse field gel electrophoresis and a southern wih your insert as probe. You should get the number of insert, location could be obtained by sequencing the fragment once cut out. Or through vectorette PCR.
Patrick

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GreenDog
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Post by GreenDog » Wed Mar 15, 2006 3:52 pm

Canalon Posted:

--------------------------------------------------------------------------------

quantitative PCR might work to give an estimate of the number of integration with the right standard.
For exact location, I would say something like, restriction followed by pulse field gel electrophoresis and a southern wih your insert as probe. You should get the number of insert, location could be obtained by sequencing the fragment once cut out. Or through vectorette PCR.


Could you eleborate more?
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canalon
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Post by canalon » Wed Mar 15, 2006 5:03 pm

qPCr or Real time PCR will quantify the amount of DNA in each PCR round. If you can compare the amount of DNA of your target and a known concentration of DNA at the same time you can deduce how much DNA you had in your sample, so if you know how muche genomes you had, you get the number of insert in each genome.

For the other one you just digest your genome in relatively large bands and run with PFGE to allow good separation of large fragments. Southern allow you to detect which fragment(s) is carrying your gene, then clone and sequence.
Patrick

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

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