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.

05/26/11 Agenda: Transforming Top 10 cells to Establish Hg sensitive promoter part K346002

(1) 5 microliters of transformation media (BIO RAD Catalogue 166-0409 received 1-17-11 GT) added to 
    40 microliters of competent cells in LB broth.

(2) 2 microliters of K346002 DNA added to broth.

(3) Solution was placed in ice bath for 5 minutes.

(4) Placed in 44.7 degree Celsius water for 50 seconds.

(5) Placed in ice bath for 5 minutes.

(6) 1 microliter of SOB buffer at rrom temperature added to broth (in order to close plasmid channels).

(7) PLated 100 microliters of cells on LB AMP plates (5-26-11 AM).

Expected: 10 to 50 colonies projected to grow.

Transformation

Decided to Transform E.col: three transformations with the E080 part, the J45004 part, and the K346002 part. We follow this procedure.
(1)105 E.col: calls were added to 100ul BioRad Transformation Solution.
(2)1ul of each part of DNA from the Registery 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 2min. Then return to ice for 30min.
(4)1ml 50B buffer 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.