From 2011.igem.org
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Week5
| Day | Note |
| Aug.1st
Monday
|
| •Fusion PCR
Vgb+YFP+tetR, NirB+RFP+tetR,
|
•PCR: NirB, Vgb |
|
| Aug.2nd
Tuesday
|
| •Cut: 13K+10I, 22M+10I
|
• Purify: 13K+10I, 22M+10I
• Ligation: Pvgb+22M+10I, Pnirb+13K+10I
|
• PCR backbones
• Electrophresis
|
• Gel excision and purification |
|
| Aug.3rd
Wednesday
|
| • Make Phusion Buffer, 5×isothermel buffer
|
• colony PCR: 20H, 20J, 22B
|
|
| Aug.4th
Thursday
|
| • Cut: 10I+22M, 10I+13; and purification
|
• Ligation: Pvgb+22M+10I, Pnirb+13K+10I,
• colony PCR: 13K+10I
|
• Culture: 20H, 20J, 22B, 1K, 1I,3C, 5E, 7C |
• Transform: 1G, 3A, 5A, 7A from the distribution plate |
|
| Aug.5th
Friday
|
| • Check the plates. Contamination, or no positive colonies |
• Miniprep: 5 backbones, 22B-1, 22B-3 |
• PCR: nirB from 13K+10I+nirB to firm the ligation. One positive result. |
• Culture the positive colony. |
|
| Aug.6th
Saturday
|
|
| Aug.7th
Sunday
|
| • Cut: 22M,13K with E & S
|
• Culture the red colonies from plate of pSB1C3 |
• PCR: 5 backbones
• Cut the PCR products with P+E and run the gel to confirm.
|
|
Week6
| Day | Note |
| Aut.8th
Monday
|
| • The gel results of 5 backbones are right.
|
• Miniprep: pSB1C3, vgb+22M+10I |
• Culture vgb+22M+1oI in hypoxia
•PCR pSB1C3
|
|
| Aug.9th
Tuesday
|
| • Run the PCR product
|
• PCR pSB1C3 again
|
• Run the product, results are good.
|
|
| Aug.10th
Wednesday
|
• Culture: 20H, 20J
• Transform 13K from plate 3
|
• Miniprep: 20H-1, 20J-1
|
• Cut: the 20H-1, 20J-1, 22B-1, 22B-3 with E+P |
|
| Aug.11th
Thursday
|
| • Run the digestion results. Bands are confirmed right.
|
• Colony PCR vgb+22M+10I
|
• Cut the plasmid of vgb+22M+10I
|
|
| Aug.12th
Friday
|
| • Make new stock of competent cells |
• Ligation: 13K+10I
• Cut the PCR results and 22M with E+P
|
• Sequence: nirB, vgbL, 12I, 20H, 20J, 22B |
• Transform 13K+10I+pSB1K3
• Cut 13K to validate. Results are right.
|
|
| Aug.13th
Saturday
|
| • Colony PCR: 13K+10I+pSB1K3 |
• Culture: vgb+22M+10I in 5ml(shaking), 5ml(water bath), 10ml(water bath), 15(water bath). |
• Check the YFP expression of 5ml(shaking) and 15ml(water bath). No yellow fluorescent cells, |
|
| Aug.14th
Sunday
|
| • Transform: the Gibson assembly results.
|
• Miniprep: 13K+10I
• Cut: 13K+10I
|
• Purification: the digestion results.
• Ligation: nirB+13K+10I
|
|
Week7
| Day | Note |
| Aug.15th
Monday
|
| • Plate: nirB+13K+10I
|
• Colony PCR: vgb+YFP+tetR +terminater |
• Gibson PCR
|
|
| Aug.16th
Tuesday
|
| • Colony PCR: vgb+22M+10I, nirB+13K+10I
|
• No bands of 13K; 22M confirmed
|
|
| Aut.17th
Wednesday
|
• Miniprep: 22M
• Cut: 22M
|
• Check CFP expression. No fluorescence.
|
|
| Aug.18th
Thursday
|
| • Transform: 13K, 10I, 22M, 12I, 18P
|
|
| Aug.19th
Friday
|
| • Miniprep: 13K, 10I, 22M, 12I, 18P |
• Cut: 13K, 10I, 22M, 12I, 18P
|
• Run the gel |
|
| Aug.20th
Saturday
|
| • Ligate 13K+10I, 22M+10I |
|
| Aug.21st
Sunday
|
| • Transform: the Gibson assembly results.
|
• Miniprep: 13K+10I
• Cut: 13K+10I
|
• Purification: the digestion results.
• Ligation: nirB+13K+10I
|
|
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