Team:ZJU-China/Notebook

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Notebook | Labnote

week1
In this week we........
week2
week3
week4
week5
week6

Week1

Day

Note

Jul.4th Monday

• Aerobic cultivation of DH5α

• Preparation of apparatus for the formation of biofilm

Jul 5th Tuesday

Jul.6th Wednesday

•Receiving primers ordered previously

Jul.7th Thursday

• Preparation of the aliquot of the primers

• Something wrong with a shaking incubator

Jul.8th Friday

• Preparation of culture plates for the transformations

Jul.9th Saturday

• Preparation of culture plates for the transformations

• protocols of transformation

Jul.10th Sunday

• Several colonies were picked up and cultivated in 5mL LB medium. •Cryosectioning of biofilm

Data

Monday

WEEK1

Tuesday

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Week2

Day

Note

Jul.11th Monday

• Set up new LB culture plates with ampicillin and kanamycin

• ·Sterilization of Glycerol and
·Preparation of 25mg/mL kanamycin

•Transformation of the parts mentioned on Jul.9th for the second time

•Observation the sections

Jul 12th Tuesday

• Pick two colonies of each parts and cultivate them in LB medium

•Transformation of three parts(20J,20H.22B from Kit plates of 2011 Distribution ) which are related to the degradation of Cellulose

•·Mini prep to isolate 10I,12I and 22M

•Conservation of 10I,12I,22M and 11P

Jul.13th Wednesday

• Place the culture plate of 20J,20H and 22B in the fridge.

•Min prep to isolate 13K •Conservation of 13K

•Restriction digest of the parts(12I,10I,22M,13K) with EcoRI and PstI

•Gel electrophoresis to analyse restriction fragments

•Test the Tm of Primers CP1&CS,NP&NS with 13K. The result of Gel electrophoresis shows that 60.2℃ is the Tm of NS and NP, and 57.4℃ is the Tm of CS and CP1.

Jul.14th Thursday

• Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers. The result of Gel electrophoresis shows that the Tm for the primers of Vgb is 54℃ and the Tm for the primers of nirB is 55℃.The RCR of YFP,RFP and tetR failed.

Jul.15th Friday

• PCR(Phusion)
• Digest 10I, 22M, 13K • Used wrong cutter, digestion again.

• Run the results of PCR and the first digestion. The annealing temperature of YFP needed change. The digestion results confirmed

• Run the digestion results of second time. The bands are confirmed.

Jul.16th Saturday

• Cut the linearized pSB1k3 with E+P

• Purify the digestion results of 22M, 13K, 10I

• Confirm digestion of pSB1k3 by electrophoresis, then purification

• Test Tm of YFP
• ligation: 22M+10I, 13K+10I

• Tm of YFP is 54 degree • transform the ligation results.

Jul.17th Sunday

• PCR 22M
• purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR.

• ligation the purified the fragments in yesterday.

• 22M PCR
• Transform the ligation results.

Monday

WEEK2

Tuesday

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Week3

Day

Note

Jul.18th Monday

• Genome extraction of E.coli

• PCR of NirB
• cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P

• Ligate: 10I+13K, 10I+22M
• Repeat NirB PCR

• Culture 11P
• Miniprep 10I, 22M, 13K

Jul 19th Tuesday

• mini-prep: 10I+13K, 10I+22M

•insert 10I+13K, 10I+22M into pSB1k3

•transform: 5E, 3C, 7C, 1K, 1I from the distribution plate

•transform 10I+22M, 10I+13K

Jul.20th Wednesday

•Colonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I confirmed

• Gibson PCR

•Colony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I

•PCR NirB

Jul.21th Thursday

•PCR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I

• Gibson PCR • Run the results of PCR verification. Bands confirmed.

• Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I • Purification of Gibson PCR results

Jul.22th Friday

• PCR amplification of Gibson assembly results

• Mini prep: 22M+10I, 22.Presever in -20
• Medi prep: 1K,1I,3C,5E,7C
• Gibson Assembly fail.

• PCR NirB by Phusion
•Repeat PCR by changing Pnibr to Gnirbr
•Cut the mini and medi prep results with E

•Run the results of PCR and digestion. Fail in PCR of NirB, succeed in 10I+3K and 10I+22M ligation.

Jul.23th Saturday

•Double digestion of 13K+10I, 22M+10I, pSB1c3

• Fail in purification of Medi prep

• PCR NirB
• Ligate NirB+13K+10I, Vgb+22M+10I

Jul.24th Sunday

• Gibson assembly: NirB+RFP+tetR
• Cut results of Gibson assembly and pSB1c3.

• Purify the ligation results in yesterday

• ligate with backbone • Culture 1I, 1K, 3C, 5E, 7C, 11P

Monday

WEEK3

Tuesday

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Monday

WEEK4

Tuesday

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Monday

WEEK5

Tuesday

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Monday

WEEK6

Tuesday

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Monday

WEEK7

Tuesday

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