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Contents 1 May 2007 1.1 May 14 1.2 May 15 1.3 May 16 1.4 May 30 2 June 2007 2.1 June 4 2.2 June 20 2.3 June 21 2.4 June 22

May 2007

May 14

1. Made the same as May 15, but did not use Type 1 (milliQ) H2O, so the solutions were discarded.

May 15

1. Made 1M CaCl2, and glycerol/CaCl2 solutions
   1. 1M CaCl2 Solution: 100mL was made
      1. CaCl2 Dihydrate = 146.986 g/mol
      2. 100mL of 1M CaCl2 requires 14.699g CaCl2 Dihydrate
   2. Glycerol/CaCl2 Solution: 30mL was made
      1. 5.00mL of 60% glycerol solution
      2. 3.00mL of 1M CaCl2
      3. 22.00mL of Type 1 (milliQ) H2O

May 16

1. Transformation of Cells:
   1. Transformed 1 vial of cells with Jun vector, and another vial with Fos vector (1uL of vector each); Sterile
   2. Used 400uL of LB (sterile) for each vial
   3. Incubated for 1 hour in shaker at 37C, then incubate overnight at 37C

May 30

1. Test PCR Machine

June 2007

June 4

1. Made Kanamycin plates
   1. 200mL of Agar
   2. 200mL of 2x LB
   3. 2mL of Kanamycin

June 20

Today: extract DNA from plates, transform, plate on amp plates.

I - Extraction of DNA from Plate (5M and 9G)

1. Puncture foil with pippette tip
2. Add 15uL of Type 1 water
3. Remove all liquid (with DNA in solution)

II - Transformation of extracted DNA

1. Chill cells on ice for 10 min
2. Heat shock cells at 42°C for 30 sec
3. Add DNA for cells
4. Add 500uL of 42°C SOC medium to each vial
5. Incubate on ice for 1 min
6. Place in 37°C shaker incubator for 1h 30min
7. Plate onto 4 separate amp plates
   1. 25uL R0062
   2. 25uL C0060
   3. 400uL R0062
   4. 300uL C0060
8. Place into 37°C incubator at 3:55PM

Next: seed, miniprep and screen the DNA.

June 21

Checked colonies in the 37°C incubator. Well formed colonies were on each plate, so plates (all 4) were transferred to the small fridge (middle shelf on the right).

June 22

Today: miniprep DNA from July 20th transformation

I - Miniprep of Colonies from R25, R400, C25, C300 (indication the biobrick and the amount in uL that was plated)

1. Transfer 1.5mL of cells suspended in LB into a microcentrifuge tube.
2. Centrifuge for 2 min at max speed (14000 rpm)
3. Remove supernatant with pipette and discard.
4. Add 250uL of buffer P1 and 250uL of buffer P2
5. Close and invert tubes 6 times
6. Add 350 uL of buffer N3 (within 5 min of adding buffer P2)
7. Close and invert 6 times
8. Centrifuge for 10 min at 13000 rpm
9. Carefully pipette off the supernatant into a Qiaprep spin column. NB: for sample R400 a large amount of supernatant was discarded. No reason, stupid move by Tim.
10. Centrifuge spin column for 60 sec
11. Discard flow-through
12. Add 750uL of buffer PE
13. Centifuge for 60 sec (max speed)
14. Add 50uL of sterile water (of EB buffer could be used)
15. Let stand for 1 min then centrifuge for 1 min
16. Transfer flow-through to a new micro-centrifuge tube (one with a cap) with a pipette.
17. Stored in -20°C freezer (green box labelled "Team 2")
18. Remaining cells in LB stored in small fridge

Next: Screen the DNA.