Help with Cloning

Prema premasundaram at hotmail.com
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
"normalizing"
restriction enzyme activities to "pmol sites per hour", and then
figure  out how much would be needed at a minimum for whatever I would
digest"

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

Thanks

Prema



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