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Today we did our first transformation via heat shock.
(1)Poured 12 LB AMP plates:(2mg of Ampicillin to 250ml of Agar)
(2)Obtained a vial of 2 microliters of TOP-10 electrocompetant E. coli cells (44-0003/793762)
(3)Added 50 microliters of pure water from the Gemini machine
(4)Added 10 ul of water to well 6E of plate 2 from the 2010 distribution, which contained part F2620.
(5)Added 2 ul of the diluted DNA in 50 ul of transformation solution (50mm CaCl2, pH 6.1 BIO-RAD Transformation Solution control # 31000008916 recieved 1-17-11 GT).
(6)Incubate on ice for 5 minutes
(7)Heat shocked at 40 degrees Celcius for 50 seconds
(8)Incubated at 4 degrees Celcius for 5 minutes
(9)Added 1mL of LB (5-17-11 DG)
(10)Plated 100 microliters and 25 microliters of transformed cells on LB-AMP (5-19-11 "crew")
We found no growth on the plates. We are going to re-transform them next week, this time using electricity instead of heat-shock.
Today we are re-transforming bacteria with the F2620, this time with electricity.
(1)Obtained 40 microliters of electrocompetent TOP-10 E. coli cells.
(2)Added 2 microliters of F2620.
(3)Zapped with 1.8kV for 5.8ms.
(4)Recovered with 900 microliters of SOB to recover cells
(5)Incubated for 1hr. Then plated the newly transformed cells.
We expect 10 to 50 colonies
Results: Lawn of bacteria!
Conclusion: No selection because AMP on plate is no good? Or maybe we had a fabulous transformation!
We replaced 100mL on LB AMP agar (5/25/2011)
We, again, expect 10 to 50 colonies
Results: No growth! This may be a result of no transformation or bad AMP on 4/21/11 plate... transformants may be present, however.
Qtip swabbed up lawn and resuspended in AMP LB broth
0 hr..................... 0.210
1 hr..................... 0.123
2.5 hr.................. 0.110
Results: absorbance declining means cells are dying... they are not transformed.
Will leave in incubator overnight to see is transformants grow out!
Next step: Try again!
So, we began another new transformation. Again, we are using heat-shock to induce the cells to take up the DNA.
(1)Obtained a vial with 40 microliters of competent cells.
(2)Added 5 microliters of transformation solution (BIO RAD, Catalog 166-0409, rec'd 1/17/11 PT)
(3)Left in ice bath for 5 minutes.
(4)Shocked for 50 seconds in 44.7 degrees Celcius water.
(5)Placed back in ice for another 5 minutes.
(6)Added 1 mL SOB media at room temperature.
(7)Plated on LB AMP plates (100 microliters of cells).
We expect 10 to 50 colonies (yet again... hopefully)
We FINALLY got growth! Last Friday, Professor Twaddle discovered our wonderfully transformed (we hope) cells. He took our best colony and placed it in growing broth. Today, we purified and isolated the DNA in order to test the transformation. We will run electrophoresis on our purified DNA next time we meet: this Thursday.
(1)We obtained 10 mL of broth containing our transformed F2620 cells. We centrifuged 2 mL four times until we had a total of 8mL worth of cells in our pellet.
(2)We added 250 microliters of resuspention solution to the pellet and vortexed.
(3)We then added 250 microliters of lysis solution and inverted. Then, we added 350 microliters of neutralization solution and inverted again.
(4)To isolate our DNA from the solution, we centrifuged for 5 minutes.
(5)Then, we transferred the lysate supernatant to our mini spin/vac column and centrifuged that for 1 minute.
(6)After decanting the flow-through, we added 750 microliters of wash solution. Then, we centrifuged again and decanted the flow-through.
(7)We centrifuged once more for 1 minute to get rid of any residual wash solution.
(8)Finally, we eluted with 50 microliters of elution solution and centrifuged.
Once we had our purified DNA, we measured the concentration. We measured it to be 31.3 ng/microliter. We stored this in the refrigerator for Thursday, when we will electrophorate it.
We decided to transform E.coli with the F2620 part as a back up.
(1)105 E.coli cells were added to 100uL BioRad Transformation Solution.
(2)1ul of F2620 DNA from the Registry was added to the cells which were incubated 30min on ice.
(3)Cells were heat shocked by swirling in water at 42 degree water bath for 2mi and then returned to ice for 30min.
(4)1ml S0B butter was added to the cells and they were incubated for 2 hrs at room temp.
(5)100ul of the transformed cells were plated on a fresh LB/AMP plate.
We have invaders! Our plates, along with three other parts from the other teams in the lab, were contaminated with fungi. So, we attempted to get some bacteria from the plates. We created a few streak plates for each part on LB AMP. We are hoping tomorrow we will have fresh, uncontaminated, transformed bacteria.
No luck. Our streak plates gave us no growth.
Conclusion: The fungus that was growing on our plates broke down the AMP, which allowed the untransformed bacteria to thrive. The bacteria we harvested, thus, were not transformed with our F2620 part.
Today, we put together our entire part. We used the A3 assembly, as explained on the Model page.
We then poured plates of our assembled plasmid. We hope for 10 to 50 colonies of transformed colonies.
Neither we nor the other high school team in the lab had growth on our transformed plates. We are going to attempt to re-assemble the part. This time, we are re-adding the correct enzymes to the already cut parts, to ensure we get enough cut. Then, we are doubling the incubation times. We are hoping the parts will thus be sufficiently cut, and the enzymes will all be killed in the heat shock.