Team:Toronto/Testing2

Wednesday, August 3

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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:
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A
B
C
D
1
SAMPLE260/280260/230CONC (ng/uL)
2
BB digested 1A-12.051.5312.4
3
BB digested 1A-22.261.039.2
4
BB digested 1B2.271.6117.7
5
BB digested 2A2.190.9411.5
6
BB digested 2B2.150.737.5
7
BB dpnI 1A2.331.1511.1
8
BB dpnI 1B1.851.0415.3
9
BB dpnI 2A2.221.1410.3
10
BB dpnI 2B2.040.9220.1
11
pCOLA 12.380.687.2
12
pCOLA 21.60.666.4
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
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Gel 1.jpg
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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

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Gel 2.jpg
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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

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Gel 3.jpg
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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