Transformation Lab

We had 4 plates that we tested. We had a controlled plate that we did not touch. I was just the plain agar. We used the LB- plain agar so we could compare and to show that even if we didn't mess with it it would still have bacteria growing in it.
The other one was LB-amp which is an antibiotic and we added the plasmid into it. The plasmid canceled the amp and as a result the bacteria grew. It was positive.
One of the other plates was LD-amp too but we did not add plasmid in it. As a result this plate did not grow at all because the amp killed it and there was no plasmid in it to block the amp from killing it. It was our only negative plate.
Out of the four plates we did have one that grew. In this plate we had LB-amp-ara. What that means is that we had LB which is agar, that is in everyone. We had amp which is the antibiotic and we had plasmid that canceled out the amp. The ara is arabinose sugar, the ara activates the genes which makes it glow under the black light.

That is what happened when we did the experiment. The steps to doing this experiment were these.
We had two little test tubes, we labeled one - and the other +; we put those back on the rack. Next we opened up the transfer pipette making sure it is kept sterile, we used that to get 250ul of transformation solution so we could put it into the - and + test tubes. We then kept the two test tubes on ice to stay cold. We used a sterile loop to pick up a single colony of bacteria from our plate. Make sure you just gently scrap it from the top, you should not cut a cube out. After we had the bacteria on the loop we put it in to the + test tube and spin the loop between our fingers until all the colonyis dispersed in the transformation solution. Make sure there is no floating chunks in the test tube. We put that test tube back in ice and got out the - test tube. With a new sterile loop we repeated the steps for the - test tube.
Next we got a new sterile loop and put it into the plasmid DNA tube, take out a loop full of the plasmid DNA. Then we mixed this plasmid with our + test tube and placed it back on ice. Do not put plasmid DNA into - test tube. We didn't because you need something to compare it to other wise they would be the same thing. After doing this you leave the test tubes in ice for 10 mins.
We labeled out agar plates on the bottom as this: LB/amp plate this +pGLO, LB/amp/ara plate this +pGLO, LB/amp plate -pGLO and last LB plate -pGLO. Step 8 is heat shock. Place the rack of both test tubes into 42 degrees C for 50 seconds. Then place both tubes back into the ice for 2 mins. After this take out the tubes and using a new sterile pipette add 250 ul of LB nutrient broth into both the tubes. Incubate the tubes for 10 minutes at room temperature. Tap tubes to mix. Using a new sterile pipette for each tube pipette 100 ul of the transformation and controlled suspensions




I really enjoyed doing this experiment. It was a very control experiment and it was nice to see what happened when you put the plates under the black light. When we got done with the experiment is when i really understood what all the step we were doing and what all the stuff we were putting in there was. I understand the steps we took and why we had 4 plates. I understand what all the amps, ara, plasmid ld stuff did kinda, for the experiment i do. I'm going to try and learn more about what all of those do with other things.