Post 3: Transformation Nightmare!!

Hi All,

You’re probably dying to know the results of the transformation reactions that I set up last post! Sorry it’s taken me so long to get back to you all!

As you can probably tell from the title, they didn’t go so well. When I very excitedly removed my samples from the incubator the following morning, I was immediately sobered. The plates were clear. There were no little white colonies. None – as in nada, zip, zero! I was flabbergasted! These TOPO-TA kits were supposed to be fool proof! And so began the tedious task of figuring out what was going wrong and why.

I set up a series of tests to try and answer this disturbing question. They are as follows:

1. A control plasmid, pUC19, was transformed into the DHa E. colicells provided in the kit on an LB plate with ampicillin.

2. Amplicons from my PCR reactions were ligated into TOPO 2.1 plasmid vectors from a TOPO kit provided by Dr. Forsyth and plated on LB+ kanamycin plates and allowed to grow overnight. This was done to determine whether or not my TOPO-TA kit was a “bad” one.

3. Amplicons from from my PCR reactions were ligated in TOPO Blunt plasmid vectors from a TOPO kit provided by Dr. Forsyth and plated on LB+ kanamycin plates and allowed to grow overnight. This was done to test the ligation of my amplicons into the plasmid vector. If the Taq Polymerase I was using for PCR was high fidelity there would be no adenine overhangs needed for ligation in my TOPO-TA kit.

4. E. coli cells from my kit were plated on LB plates with no antibiotics. This was done to test the viability of the cells in my kit.

The results were in the next morning. The cells with no plasmids grew up beautifully. Thus, my cells were viable. The  plate with the blunt TOPO vectors showed no colonies. My PCR amplicons were not being blunted by the Taq Polymerase I was using. The control plate also showed no colonies. This was disturbing. This could mean my TOPO-TA kit came from a bad lot. This hypothesis was confirmed by the presence of more than 100 little white colonies on the TOPO-2.1 plate! MY transformations were working! My kit was bad.

Needless to say, we ordered a new TOPO-TA kit. My transformations have been working beautifully since! Stay tuned to follow the bacterial DNA on to the next step: Mini-Prep Mayhem!

Dana

P.S. Much thanks to Dr. Forsyth for helping me uncover what was going wrong with my transformations!