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From 2011.igem.org

8/1
- Digest:
o OR (E +S)
o 34 Cro (E +X)
- Grow up:
o 34 TEV + B0015
o K3
- Insert PCR:
o OR + 34Cro
- PCR:
o C0051 + B0030 (R)
o C0051 + B0031 (R)
o C0051 + B0034 (R)
 0
 1
 4
- Primers used:
o 0A
o 1A
o 4A


8/2
- Digest:
o Or+34 Cro -> E + S
o 34 EV + B0015 (vector) -> E + X
o K3 (vector) -> X + P
o B0030 + C0051 -> X + P
o B0031 + C0051 -> X + P
o B0034 + C0051 -> X + P
o Or + K648000 -> E + X (vector)
o B0030 + C0051 -> E + S
o B0031 + C0051 -> E + S
o B0034 + C0051 -> E + S
- Ligated and Plated:
o B0030+C0051+K3
o B0031+C0051+K3
o B0034+C0051+K3
o Or+34 Cro+34 TEV+B0015 (A)
o Or+34 Cro+34 TEV+B0015 (B)
o B0030+C0051+Or+K648000
o B0031+C0051+Or+K648000
o B0034+C0051+Or+K648000


8/3
- Colony PCR:
o B0030+C0051+K3
o B0031+C0051+K3
o B0034+C0051+K3
o Or+34 Cro+34 TEV+B0015 (A)
o Or+34 Cro+34 TEV+B0015 (B)
o B0030+C0051+Or+K648000
o B0031+C0051+Or+K648000
o B0034+C0051+Or+K648000
o B0030+C0051+K3


8/4
- Minipepped an sent to Sequencing:
o B0034+C0051+Or+K648000 (R, F)
o B0030+C0051+K3 (B)
o B0030+C0051+K3
o B0030+C0051+Or+K648000 (F, R)
o B0030+C0051+Or+K648000
o B0034+C0051+K3
o Or+34 Cro+34 TEV+B0015 (B)
o B0021+C0051+K3
o Or+34 Cro+34 TEV+B0015 (A)
o Test A
o Test B
- Digest:
o B0030+C0051 -> E + S
o B0031+C0051 -> E + S
o B0034+C0051 -> E + S
o Or+34 Cro+34 TEV+B0015 -> (vector) E + X
- Ligation:
o B0030+C0051+Or+34 Cro+34 TEV+B0015 (A + B)
o B0031+C0051+Or+24 Cro+34 TEV+B0015 (A + B)
o B0034+C0051+Or+34 Cro+34 TEV+B0015 (A + B)
- Tranform
- Stock = (Done)
o B0030+C0051+K3
o B0031+C0051+K3
o B0034+C0051+K3


8/5
- Digest:
o Or+34 Cro -> E + S
o 34 TEV + B0015 -> E + X (vector)
o K3 -> X + P (vector)
o B0030+C0051 -> X + P
o B0031+C0051 -> X + P
o B0034+C0051 -> X + P
o Or + K648000 -> E + X
o B0030+C0051 -> E + S
o B0031+C0051 -> E + S
o B0034+C0051 -> E + S
- Ligation:
o B0030+C0051+K3
o B0031+C0051+K3
o B0034+C0051+K3
o Or+34 Cro+34 TEV+B0015
o B0030+C0051+Or+K648000
o B0031+C0051+Or+K648000
o Or+34 Cro+34 TEV+B0015
o B0030+C0051+Or+K648000
o B0031+C0051+Or+K648000
o B0034+C0051+Or+K648000
- Transformed and Plated – all the above


8/7
- Grew colonies from all that was plated on 8/5
8/26
- Digest: (R=Reverse)
o Or+34 Cro+34 TEV+B0015 -> E + X -> Phosphatase
o B0030+C0051 -> E + S
o B0031+C0051 -> E + S
o B0034+C0051 -> E + S
- Ligated, Transformed, and Plated:
o B0030+C0051+Or+34 Cro+34 TEV+B0015
o B0031+C0051+Or+34 Cro+34 TEV+B0015
o B0034+C0051+Or+34 Cro+34 TEV+B0015


8/28
- Grew up:
o B0030+C0051+Or+34 Cro+34 TEV+B0015
o B0034+C0051+Or+34 Cro+34 TEV+B0015


8/29
- Miniprep and Sequence:
o B0030+C0051+Or+34 Cro+34 TEV+B0015
o B0034+C0051+Or+34 Cro+34 TEV+B0015
- 1:1 Molar Solution:
o 660 per base


9/9
- Digest:
o B0030+C0051 -> X + S
o B0031+C0051 -> X + S
o B0034+C0051 -> X + S
- Ran a gel:
o Ladder
o B0030+C0051
o B0031+C0051
o Or+34 Co+34 TEV+B0015
o B0034+C0051
- Extracted the DNA from gel and nanodropped before ligating


9/16
- Miniprepped and PCR’d:
o B0030+C0051 (Primers used -> 17679 + 17680)
o B0031+C0051 (Primers used -> 17681)
o B0034+C0051 (Primers used -> 17682)
- Tecan testing Or
o Or Mcherry 1.71 -> 6µL
 D 2 3 4
o Or K648000 1.44 -> 7µL
 E 2 3 4
o Or K648000 1.02 -> 10µL
 F 2 3 4
o B2 .40 -> 25µL
o B4 .8 -> 12 µL
o B6 .65 -> 15 µL
o D2 .7 -> 14 µL
o D4 .8 -> 12 µL
o D6 .75 -> 13 µL
o F2 .4 -> 11 µL
o F4 .94 -> 11 µL
o F6 .9 -> 11 µL

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