gel extraction problems with the new kit from Macherey&Nagel - (Nov/14/2012 )

Since Macherey&Nagel has changed their NucleoSpin Extract II kit, we have problems cloning. We have isolated the problem to the gel extraction part. Did any of you observe any problems there?

Andreea

I'd like to convince you to avoid gel purification. Why are you doing it?

weeell: I have been having the same thoughts lately. However, since I am kind of the person figuring this thing out for the entire department, let us not have to explain to 30 people that the cloning how they used to know it is wrong
So a classical cloning protocol in our lab:
1 - PCR insert + insertion of restriction sites + DpnI digest ON + PCR clean-up kit + RE 15 min to 2 h + inactivation + PCR clean-up
2- Midi prep the vector + RE as above + inactivation + gel extraction
3-ligate with T4 DNA ligase for 15 min to 1 hour at RT or ON at 16oC
4-transform in XL1Blue

I have tested everything. Let's assume that I have huge amounts of evidence that it is the gel extraction step aka all the people that had vectors digested in the freezer have successful cloning; people who do Topo or Gateway cloning as well. But for specific vectors you really have to use the classical way.

Andreea

I would do this with this technique:
1) PCR insert, Ampure bead cleanup, RE digest (with DpnI), heat kill
2) PCR vector, Ampure bead cleanup, RE digest (with DpnI), heat kill (this can be done ahead of time, carefully)
Choose a vector with different resistance than your insert. Possible if you have a broad range of prepared vectors.
We have our standard vectors in amp, kan, tet, chlor.
3) Ligate 15 min on the bench.
4) Transform to DH10B

For some reason, people think there is a reason not to PCR vectors. But it is by far the easiest and most reliable way to prepare low-background vector backbones. Usually, you don't even care about the accuracy of the vector -- if it conveys resistance and carries your insert, you're fine.

Note that this scheme works well for multi-part cloning for making more complex assemblies.

Just say no to gels and columns!

I did this for exactly what you said: multi-part cloning e.g. producing new vectors; but now I really have to make it possible for the people to use the old fashion way that used to work. And I am wondering whether is the kit (which would be such a not ok thing) or the gel staining bath- we use Gel Green. It might be the bath since no matter how many times I try to explain to people not to leave gels overnight or to refresh it regularly, they do not get it.

For myself, I can do what you suggested. However, for some of the clonings I do at the moment I cannot have another resistance vector. I thought about this; or ccdB or sacB... but for these insect cell expression vectors I do not have another alternative other than insert from Amp template to Amp vector.

Andreea

It can pay off rapidly to engineer a few different resistance plasmids. Your cloning can be done in any vector with any resistance, and you can transfer to an amp vector in a final stage (using the PCR technique).