From 2011.igem.org

Revision as of 21:32, 26 May 2011

May

May19

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, 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")

May 18

We found no growth on the plates. We are going to re-transform them next week, this time using electricity instead of heat-shock.

May 24

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

May 25

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

May 26

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
Time....................A600
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, tec'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)