Team:ZJU-China/Notebook/August

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Labnote

This group...........

Week5


DayNote
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


DayNote
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


DayNote
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

 

Week8


DayNote
Aug. 22nd Monday
• Transform 13K+10I, 22M+10I • Test new primers.
• PCR: 22M+10I, 13K+10I
• Run the PCR products.
• Cut the PCR products.
Aug. 23rd Tuesday
• Test primers: CS/CP, VF/VR, pSB1_f/r • Cut: 11P, 1F, 22M
• Run the cut results.1F-1/2/3 are right.
• Mix the 1F-1/2 purification products • Cut: 1F-1+1F-2(E+S), 22M-3(X+P), pSB1C3(E+P)
Aug. 24th Wednesday
Aug. 25th Thursday
Aug. 26th Friday
• Amplify: vgb, 1C3
Aug.27th Saturday
• Digest: Vgb, 1C3, fdfhF, 22M+10I, 13K+10I
Aug.28th Sunday
• Ligate: vgb+22M+10I, fdfhF+13K+10I • Transform the ligation results.