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From 2010.igem.org

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Contents 1 July 2010 1.1 July 5/2010 1.2 July 5/2010 Evening 1.3 July 6/2010

July 2010

July 5/2010

(In Lab: JV, AV, HB)

Objective: Run a 1% agarose gel of purified PCR samples from June 24/10

Method:

Lane	Sample	Components (µL)
1	1kb Ladder	0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
2	1 - pBAD (A4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
3	2 - pBAD (A5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
4	3 - SRBS (A6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
5	4 - SRBS (A7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
6	5 - CFP Complete (A8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
7	6 - SRBS (A10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
8	7 - EYFP (B1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
9	8 - N term tag (B2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
10	9 - pSB NEYFP (B4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
11	10 - pSB NEYFP (B5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
12	11 - CFP (B6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
13	12 - pBAD-TetR (B10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
14	13 - D3	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
15	14 - C term (D4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
16	15 - C term (D5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
17	16 - pLacI (D6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
18	17 - NEYFP (E2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
19	18 - CEYFP (E6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
20	19 - CEYFP (E7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1	1kb ladder	0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
2	20 - EYFP (E8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
3	21 - EYFP (E9)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
4	22 - EYFP (E10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
5	23 - ECFP (F1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
6	24 - ECFP (F2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
7	25 - ECFP (F3)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
8	26 - pBAD-TetR (F4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
9	27 - pBAD-TetR (F5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
10	28 - EYFP (G1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
11	29 - pSB CEYFP (G4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
12	30 - pBAD (1) (G6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
13	31 - pBAD (2) (G7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
14	32 - N term tag (G8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
15	33 - lumazine (G9)	1 DNA + 2 Dye (6X) + 7 Milli-Q H2O

Ran gel at 100V for 45 minutes.

July 5/2010 Evening

Objective:To over-express CFP complete in DH5α

Method:

1) Inoculated 6mL culture with ampicillin and glycerol stock A6-May 13,2010 CFP complete 2) Went in the shaker at 6:50pm

July 6/2010

(In lab: JV, AV, HB)
Objective:To continue the over-expression of CFP complete in DH5α

Method:
1) Put both cultures (taken out of shaker at 9:15am) and put them in 500mL of LB w/ Amp.
2) initial OD was 0.071 (600λ)

Issue:

• After checking the the sequencing it was evident that our promotor is always off. It is turned off by the product of the gene TetR. Which is not part of our construct.
  

table>OD (600λ)

Time (hours)
0	0.071
1	0.390
1.5(T0)	0.606
2.5 (T1)	1.250
3.5(T2)	3.04
4.5(T3)	2.75

Results:Ran samples on a 15% SDS-page. The gel did not show any signs of over-expression.