digest, blunt end, religate, digest.... - so many steps, maybe can skip purification... (Sep/26/2006 )
this is really complicated. my freind has given me wrong idea about the cloning sites of the vector that she has, i designed PCR product according to that, and now it comes out that the whole MCS is cut out by her and she only has the original received plasmid with the insert (her protein) and MCS intact.
so now i have to:
1-cut out her insert
2-blunt end (klenow)
4-electroporate, miniprep colony.
5-digest with proper enzymes
6-ligate with my insert.
i usually digest and purify by cutting out of low melting temp gel. but I phenol-chloroform after that. however if I do all phenol-chloroform steps i will lose much of DNA, sicne they are too many. where can I skip them????
If you gel purify after the digest, and use a gel purification kit (etc Qiagen) to remove the gel matrix (no enzymes like B agarase used) you do not need to do a phenol-chloroform extraction. Most of the larger protein junk will be trapped in the gel matrix or washed away during the column binding.
However if the above suggestion is not applicable for one reason or another, a simple suggestion would solve the problem.
Midiprep (100ml) the miniprep!
Midiprep the cell containing the right construct and digest buckets of plasmid DNA! That way there is no fear of losing DNA because there is so much you can afford the loses.
P.S: Is there anyway possible to do a colony PCR to screen the colonies?
Catch line to convience the doubting supervisor:
It is faster (1 day oppose to 2 days), less time consuming(2 hrs verse 1 day), able to screen far more colonies (with multichannel pipette + 96 well PCR plate + 96 well micotiter plate)(48 vs 96) and on the whole cheaper.
And all my friends do it too.
Can you PCR amplify the insert, and clone it into a TA-cloning vector? Then you'd have it in an intact MCS, and would be able to liberate it by digestion with enzymes leaving with proper ends for your ultimate vector...