Team:ZJU-China/Notebook

From 2011.igem.org

Revision as of 06:32, 3 October 2011 by Tlytiger (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">

Labnote

Biobrick group | Biofilm group

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

July

In this month we......
>>Click to see

August

In this month we......
>>Click to see

September

In this month we......
>>Click to see

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

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.

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

Week4

Day

Note

Jul.25th Monday

• Transform Pnirb+13k+10I+pSBK3-2

• Place the culture in 4 degree

• Medi-and mini-prep of five cultre

•Gibson Assembly confirmation by gel

Jul. 26th Tuesday

• Cut Pnirb, Pvgb,(E+S)
• Cut 10I+13K, 10I+22M.(X+P)

• Ligation: vgb+10I+22M, nirB+10I+13K,

• Ligation: pSBK13+vgb+10I+22M, pSBK13+nirB+10I+13K

• Transform the ligation results.

Jul.27th Wednesday

• No colony on the plate
• Gibson assembly

• Run the results of Gibson assembly

• Excise the bands, the purify the DNA
• Gibson Assenmly

Jul.28th Thursday

•2011 China Meet-up @ Hefei

Jul.29th Friday

•2011 China Meet-up @ Hefei

Jul.30th Saturday

Jul.31th Sunday

• Culture 22M+10I, 13K+10I

• Cut a0H, 20J, 22B

Data&protocol

Jul.13th Wednesday

Systems of restriction digestion with EcoRI and PstI

Plasmid	1μL (>100ng)
EcoRI	1μL
PstI	1μL
10×Buffer Tango	2μL
ddH2O	15μL

Temperature grad
56℃,57.4℃,60.2℃,62.9℃,64.3℃,65.8℃

PCR system (test the Tm of the primers CP1&CS, NP&NS)

10×Buffer	2μL
dNTPs	0.5μL
primers CP1 (NP)	1μL
primers CS (NS)	1μL
Template	1μL
rTaq	0.2μL
H2O	14.5μL
Total	20μL

Jul.14th Thursday

PCR systems(test the Tm of the primers for YFP,RFP,tetR,Vgb and nirB )

10×Buffer	2μL
dNTPs	0.5μL
primers F	0.5μL
primers R	0.5μL
Template	1μL
Taq	0.2μL
H2O	15.5μL
Total	20μL

Temperature grad and the results:

vgb&nirB pcr test:

Early July:

Pre-experiments with biofilm formation with circular and non circular silicone tube, 24 well plate on different support including glass, paper, plastic film, rubber. The final material are used based on the easiness biofilm form on them and on the easiness to observe under microscope.

28th July:

13.30: DH5ɑ 11p 5mlX4
23.00: silicone tube set at 37℃

29th July:

13.00: LB culture 50ml with circular culture medium. LB culture 50ml with noncircular culture medium.

31st July:

13.00: -80℃ storing silicone tube. A thick white and loosely bond substance is seen on the inner wall of the 5mm silicone tube, and on the inner wall of the 1mm silicone tube a flatter and smoother white substance. Especially obvious where the tube turns. Possibly because the speed of culture flow is slower.