DH10B with big construct question - (Aug/15/2012 )

Hello: I am trying to clone a 2.5 kB insert with different sticky ends (Bam H1, Spe1) into a large vector (pGL3 with other fragments after the Luc sequence), the vector is 10 kB, so the total size is 12.5 kB The ligation product is being electroporated into DH10B cells, and then plated on Amp resistance plates (Amp = 100 ug/mL).

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.

I would use less culture in your pcr reactions -- probably dilute it in water first. You don't have to wait for outgrowth overnight. You can do colony pcr directly from the plate by touching a tip to the colony, putting the tip into 50 ul of water and scraping it, then using the same tip to put 4 ul of the water onto a master plate and inoculate an overnight (optional). 0.5 ul of the water is used in a colony pcr reaction with primers: one to the vector backbone, another to the insert. There is some danger of false positives with this approach, but it saves a day or so.

Concerning the fact that some of your plasmids are smaller than expected:

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).