Is ligation really necessary?
Posted 05 April 2013 - 04:39 PM
Posted 05 April 2013 - 07:44 PM
Would you recommend PEG for restriction ligations or is this usually included in buffers ( I used NEB T4, btw)?
Edited by Ahrenhase, 05 April 2013 - 07:48 PM.
Posted 06 April 2013 - 08:21 AM
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Posted 06 April 2013 - 12:48 PM
Posted 06 April 2013 - 03:55 PM
You might consider making vector with PCR rather than by cutting it. The major difficulty with cutting vector is that some of it fails to cut. You can reduce the problem by linearizing with PCR instead cutting with a RE. You can remove most of the template DNA with a DpnI digest. You can test for background with a vector only ligation control.
Posted 06 April 2013 - 04:30 PM
I'm using colony PCR now for speed, but I am using primers that flank the cloning site. So a hit should pop up. When I first found inserts, I grew up cultures and used 1 uL of a turbid culture for the PCR. I don't think there should be much difference between these 2 methods with reference to the results.
PCRing my vector wouldn't be easy, I don't think. It's 6kb long and we don't have any of those fancy lambda phage polymerases.
Edited by Ahrenhase, 06 April 2013 - 04:37 PM.