Help with Cloning

Prema premasundaram at
Wed Sep 29 23:46:47 EST 2004

Thanks for replying. Here are the clarifications you needed.

1. how big is the insert relative to the vector?
The insert is 2kb and vector is ~2700bp

2. how much are you digesting?
I did a miniprep and digested around 5 ul of DNA in a final volume of
20ul. I did not spec the DNA for concentration. Also the miniprep did
not include RNase treatement. I use 1ul enzyme and usually digest for
2 hrs.

3. how much of your digest are you loading onto your agarose gel?
I load all the 20 ul on the gel to cut out the bands. 

4. how do you test the identities?
I construct my plasmid maps on DNA strider and choose enzymes that
will confirm the presence of the insert in the desired vector
background. I then miniprep the transformed colonies and digest with
the chosen enzymes, run a gel to see if I get the right size bands for
the clone I am looking for. That is when I found the digests look like
the transformants I got all contained one of the parent plasmids that
I used.

5. Yes. I do know that KpnI and NotI don't work in the same buffer. I
use NEB enzymes and buffers. I digested with KpnI first, run it
through a qiagen column, elute in 30ul H2O and then digest with NotI
by adding its buffer.

6. "P.S. what I tried to do early on is to get in the habit of
restriction enzyme activities to "pmol sites per hour", and then
figure  out how much would be needed at a minimum for whatever I would

With reference to the above suggestion could you tell me how to
normalize. I am new to the cloning techniques.



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