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Team:SouthBend-Mishawaka-HS-2/Notebook
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Notebook
05/19/11 Agenda:Transformation Test Run
(1) Obtained a vial of 2mL of TOP-10 electro competent E. coli cells 3-16-11 (44-0003/793762)
(2) Added 50 microliters of BIO-RAD transformation solution (Control number 31000008916 recd 1-17-11 GT)
(3) Added 2 microliters of DNA from Registry plate 2 well 5b
(4) Incubate on ice 5 minutes.
(5) Heat shocked 40 degrees C for 50 seconds
(6) Incubated 4 degrees C for 50 seconds
(7) Added 1mL LB 5-17-11 D.G.
(8) Plate 100 microliters and 25 microliters of transformed cells on LB-AMP (5-19-11 "who")
05/24/11 Agenda: Establishment of the K346002 Hg Sensitive Promotor
(1)Electroporation of K346002 into Top 10 cells.
(2)40 microliters of Top 10 cells (JH 5-23-10)10E9 + 2 microliters K346002 DNA from Registry.
(3)Parameters of electroporation: not recorded.
(4)Added 1 microliter of SOB media (JH 2-11-11)immediately after electroporation.
(5)Incubated tube at 37 degrees Celsius for 30 minutes.
(6)Plated 200 microliters of transformation mixture on LB Agar AMP 5 micrograms/microliter (4-21-10). Stored mixture at 4 degrees Celsius.
Expected: 10 to 50 colonies projected to grow.
05/25/11 Agenda: Examining Results of Establishment of the K346002 Hg Sensitive Promotor
(1) Examined results from yesterday: Lawn of bacteria!
(2) Conclusion: No selection either due to inconclusive AMP or fabulous transformation.
(3) Replated 100 microliters K346002 on LB AMP Agar (AM 5-19-11)
Expected: 10 to 50 colonies projected to grow.
05/26/11 Agenda: Examining Results of Establishment of the K346002 Hg Sensitive Promotor Round II
(1) Results: NO GROWTH!
(2) Conclusion: Perhaps no transformation and bad AMP on 4-21-10 plate. However, could still be some transformants present.
(3) Q-tip swabbed up lawn and resuspended in AMP LB broth. Resuspension fluid placed in photospectrometer.
(4) Photospectrometer results for K346002: Time A600 nm 0 hr. .315 1 hr. .148 2.5 hr. .133 (5) Results: absorbance is declining meaning cells are dying, thus no transformation has resulted. Will leave in incubator overnight to see if transformants grow out.
