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console.js:26

Wednesday, August 3

Wednesday, 8/3

Members Present:

LAB:

Morning:

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Miniprep of lacZ ligation O/N cultures

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Nanodrop of miniprepped ligations:

	A	B	C	D
1	SAMPLE	260/280	260/230	CONC (ng/uL)
2	BB digested 1A-1	2.05	1.53	12.4
3	BB digested 1A-2	2.26	1.03	9.2
4	BB digested 1B	2.27	1.61	17.7
5	BB digested 2A	2.19	0.94	11.5
6	BB digested 2B	2.15	0.73	7.5
7	BB dpnI 1A	2.33	1.15	11.1
8	BB dpnI 1B	1.85	1.04	15.3
9	BB dpnI 2A	2.22	1.14	10.3
10	BB dpnI 2B	2.04	0.92	20.1
11	pCOLA 1	2.38	0.68	7.2
12	pCOLA 2	1.6	0.66	6.4

Miniprepped Ligations (LacZ + pCOLA)

The two types - BB digested and BB dpnI, refer to wether or not the pSB1C3 backbone was PCR amplified from RFP , or was further gel extracted and dpnI digested. pCOLA refers to the construct pieces given by Kayla as a positive control. 1/2 refers to which plate they were taken from, as two of each transformant were made. Since two colonies were selected from each plate, A + B distinguishes the two.

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RE single digest of miniprepped lacZ ligations, as well as Kayla's pCOLA ligations

Afternoon:

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Made more rubidium chloride competent cells (both DH10B and BL21), labelled CCE and CC Bl21, respectively

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Ran gradient PCR on Long GolS (G 1-6) , Long GolS P118A (P 1-6), mCherry (C 1-6).

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Ran 3x15 lane gels on RE digested lacZ constructs, pCOLA, and on PCR gradients

Gel 1.jpg

Lane 1: BB digested 1A-1 Lane 2: BB digested 1A-2 Lane 3: BB digested 1B Lane 4: BB digested 2A Lane 5: BB digested 2B Lane 6: 2 log ladder Lane 7: dpnI digested 1A Lane 8: dpnI digested 1B Lane 9: dpnI digested 2A Lane 10: dpnI digested 2B Lane 11: 2 log ladder Lane 12: pCOLA 1 Lane 13: pCOLA 2

Gel 2.jpg

Lane 1: G1 Lane 2: G2 Lane 3: G3 Lane 4: G4 Lane 5: G5 Lane 6: G6 Lane 7: 2 log ladder Lane 8: P1 Lane 9: P2 Lane 10: P3 Lane 11: P4 Lane 12: P5 Lane 13: P6 Lane 14: 2 log ladder

Gel 3.jpg

Lane 1: 2 log ladder Lane 2: C1 Lane 3: C2 Lane 4: C3 Lane 5: C4 Lane 6: C5 Lane 7: C6 Lane 8: 2 log ladder

TO DO:

For the next day:

LAB TEAM:

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PCR of backbone

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PCR purification of backbone and constructs pcr amplified today - GolS, p118A, mCherry)

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Vacufuge some of the larger constructs to an appropriate conc for digest (ask andy for advice)

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RE digest of constructs and backbone

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rSAP of backbone

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PCR purification

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Ligation O/N at 16C

LAB MANAGERS:

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Some autoclaving would be noice