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Different ways to ensure the successful cloning?


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#1 tmaz

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Posted 04 December 2013 - 07:14 AM

Hi,

 

I'm cloning a digested insert to the digested vector (cut with same two restriction enzymes) which harbors Ap resistance. After transformation, is there any other way to confirm that I have a insert + vector clone than sequencing the purified plasmid or amplifying the insert with spesific primers?

 

I'm asking this because I assume that there will be also transformants on the Ap plates with the plain linearized vector which lacks the insert. The insert itself doesen't carry any selective marker. I have purified the linearized vector from agarose gel but I guess that I can't be sure that the sample contains only linearized form..?  I don't want to proceed to the next step of the work unless I'm sure that I have a "right" transformant.  

 

Thanks!


Edited by tmaz, 04 December 2013 - 07:16 AM.


#2 phage434

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Posted 04 December 2013 - 07:46 AM

If you do a miniprep and cut your DNA with the two REs, then you should see the insert length band on the gel. You can also PCR using one primer on the insert and a second on the vector. You can even do this PCR directly on a picked colony (see "colony PCR" for a protocol). The main trick is to pick very tiny amounts of the colony into 50 ul water, mix, and use 1 ul of that water in a PCR reaction. Extend the initial 95 denaturing cycle to 3 minutes. You should capture the colony by spotting 4 ul of the water onto a selective plate in grid form (a multichannel pipettor can be used here).



#3 ostiaziocan

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Posted 04 December 2013 - 07:54 AM

Here's a clear explanation about double digestion to detect plasmid inserts smile.png

 

http://www.methodboo...a/restrdig.html






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