TA cloning results

Yesterday I transformed E.coli with the ligation product of TA cloning. The results look good! All the controls worked as expected.

The CFU for my Rec1 experimental batch were 4*10^8, and the transformation efficiency was 1.15*10^-5,  plenty of white colonies to pick from. There are some small white colonies on most plates that look very distinct, they aren’t fuzzy and have a very flat white texture(see pic 1). I believe these are contaminants as they showed up in the no DNA controls. They are rare, making up around 5% of white colonies.

I plated on X-gal(for CFU) and X-gal+Ampicilin(For Transformants) LB plates. No IPTG was used. The controls I did are as follows:

“- Control” Ligation with vector but no insert DNA. Expected to see all blue colonies on ampicilin plates, found 44 blue colonies and 1 white colony, although it is a contaminant type (see pic 2)

“+ control” Ligation with vector and control insert DNA. Worked as expected, had lower CFU but higher TF than the rec1 plasmid

“No DNA control”  E.coli transformed with no DNA. I expected not to find any colonies whatsover on AMP plates, I found a single colony with a blue center and white outside(see pic3).

“Positive (plasmid) control” E.coli transformed with an equal molarity of an uncut vector containing AMP-r but not LacZ. I expected to see only white colonies on Amp plates and I did, although one plate had a little areas that looked like galaxy, white colonies diminished to the smallest visible size accumulated in a patch. I can’t explain this. The other identical plate was cleaner, but had a far lower TF than rec1. Also two very large colonies with blue centers appeared on the CFU plates. I can’t explain that either(contam?).

“Heat shock control” Same as No DNA control, only spent 50s at room temperature instead of the 42 degree bath. No transformants, very slightly lower CFU than No DNA control(CFUs were similar among all treatments)

Pic 1: Contaminant white colony. From plate 1, a rec1 CFU plate with x-gal but no Ampicilin.

 

 

Pic 2: The single white colony on the – control, ampicilin+x-gal plate

 

Pic 3. A lonely colony that is blue in the center and white on the outside that showed up on a x-gal/Amp no DNA control.

 

I re-streaked 10 white colonies from the Rec1 plates on X-gal/Amp LB plates and put them in the incubator, along the smaller white “contaminant” colony from pic 1. I’ll pick a colony from each restreak, and use the same colony to both inncolate broth and perform PCR to test for Rec1. Afterwards I’ll digest the strains that had the proper PCR amplification for rec1. I put the initial plates into the fridge, just incase none of the ten colonies I restreaked work I can always go back to them and pick new ones.

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