gel extraction problems with the new kit from Macherey&Nagelgel extraction
Posted 14 November 2012 - 02:18 PM
Posted 14 November 2012 - 02:21 PM
Posted 14 November 2012 - 02:28 PM
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.
Posted 14 November 2012 - 02:49 PM
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!
Posted 14 November 2012 - 02:57 PM
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.
Posted 14 November 2012 - 05:54 PM