Posted 15 August 2012 - 05:40 AM
1.) I dont have blue white screening with this insert and the pGL3 plasmid. Instead, what I do is pick a colony, create a 1 mL LB overnight, test 2 uL of this with 13 uL qPCR mix with primers specific for the insert (amplicon is 300 bo). I have to check a lot of colonies (like 50 or more). From many colonies/growths I get a positive, but weak PCR amplifcation. I will miniprep this growth and see a contaminating plasmid. Is there a better way?
2.) I get few colonies as I expected, but the colonies seem all seem to be derived by a contaminating plasmid (this appears to be 2 or 3 kB) in size. Has anybody else have this problem? How can I solve it?
3.) Im guessing that my transformation effiency for this large construct (12.5 kB) is low. Is there anyway that I can increase this?
Thanks for any consideration.
Posted 15 August 2012 - 05:54 AM
Posted 20 August 2012 - 02:24 AM
I have used DH10B for electroporating ligations of plasmids larger than 14Kb, and in my experience, it is normal not to have many many colonies.
In some occasions, what I have seen is that when I pick the colonies to be tested just after 1 night at 37 ºC, most of the colonies contain plasmids that are much smaller than expected, and only a very few are positive. However, after leaving this same plate at 4 ºC in the fridge, I observed that new small colonies have started to grow and most of them are now positive for my expected plasmid.
Leaving the plate for a longer time at RT or 4 ºC, may be important if you need more than one positive colony.
And of course, electroporation can be optimised in several ways (do not wait more than a few seconds after the pulse before adding SOC, for example).