Team:UT-Tokyo/Notebook

From 2012.igem.org

Notebook

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Correspondence table

No. Biobrick Parts
#1 pConst
#2 Argbox
#3 RBS
#4 Argbox×8
#5 d.term
#6 fake of fhlA
#7 fhlA(P-siteless)
#8 hycAp
#9 Ptrc-RBS-rocF
#10 fhlA(E363G)
#11 RFP(psb1K3)
#20 pLac
#40 RBS(weak)-GFP-d.term
#44 RBS(strong)-GFP-d.term
#66 RFP(psb1C3)

Lab Notes

May

1st week: 5/27-6/2


May 29th

  • Making LB medium

May 30th

  • Making LB Agar
  • Making LB Amp Plates


May 31st

  • Preculture


June

2nd week: 6/3-6/9

June 5th

  • Plating JM109

June 6th

  • preculture

June 7th

  • Measuring OD by spectroscope
  • Meking electrocompetent cells
  • Electroporating of a test plasmid

June 8th

  • Caluculatiing transformation efficiency


3rd week: 6/10-6/16

none

4th week: 6/17-6/23

none

5th week: 6/24-6/30

June 26th

  • Making LB tetracycline plate

June 27th

June 28th

  • Making LB kanamycin plate and chloramphenicol plate
  • Transformation with a test plasmid

June 29th

  • Transformation with #1(constitutive promoter) and #11(RBS)

July

6th week: 7/1-7/7

July 3rd

  • Making K solution
  • Colony PCR DNA fragment assumed as fhlA (#6) for cloning

July 4th

  • Miniprep #1, #11

July 5th

  • Making 1% agarose solution
  • Electrophoresis (#6)
  • Digest (#1_EP, #11-_EP)

July 6th

  • Colony PCR (#6)

7th week: 7/8-7/14

July 9th

  • Electrophoresis (#6)

July 10th

  • Transformation
    • #3(RBS medium), #5(d.term), #20(pLac)

July 11th

  • Preculture
    • #3, #5, #20
  • Gel Extraction(#6)

July 12th

  • The continuation of Gel Extraction(#6)
  • Miniprep
    • #3, #5, #20

July 13th

  • Nanodrop (#6).
  • Digestion (#3:E-P, #5:E-P, #20:E-P)
  • Electrophoresis (#3, #5, #20)

July 14th

  • Miniprep (#1,3,5,11,20)
  • Gel extraction (#6)

8th week: 7/15-7/21

July 17th

  • Transformation (#1A2,#3,#5,#20)
  • PCR (parts of 1. and #3,#20(Colony PCR))
  • Incubate #5.

July 18th

  • Miniprep (#1A2,#3,#5,#20)
  • PCR using u.p (#1A2,#3,#5,#20)
  • Colony PCR (#3,#20,H₂O)
  • Electrophoresis (#1A2,#3,#5,#20, #3,#20, H₂O)
  • PCR (2011’s #1,#36 and 2012’s #3,#5,#20, H₂O)

July 20th

  • Miniprep.
  • Electrophoresis (7/19’s PCR product(#1,#3,#5,#20,#36) )
  • Colony PCR (#3,#5,#20)

July 21st

  • Electrophoresis (7/20’s PCR product (#3,#5,#20))


9th week: 7/22-7/28

July 24th

  • Making agar medium plate contain ampicillin
  • PCR (#6, #8)
  • Electrophoresis (#6, #8)
  • Transformation (#1)
  • Culture (#5, #20)

July 25th

  • Miniprep (#5, #20)
  • Purify (#6, #8)
  • Digestion (#3:S-P,#5:E-X,#11:E-S,#20:S-P)
  • Electrophoresis (#5, #11)
  • Ligation (#5,#11) (?)

July 26th

  • Culture (#1)
  • Digestion (#3:S-P, #5:E-X, #11:E-S, #20:S-P, fhlA:E-S, hycA:E-S)
  • Electrophoresis (#3:S-P, #5:E-X, #11:E-S, #20:S-P, fhlA:E-S, hycA:E-S)
  • Gel extraction (#3:S-P, #5:E-X, #11:E-S, #20:S-P, fhlA:E-S, hycA:E-S)

July 27th

  • Culture, Miniprep (#1)
  • Digestion (#5-A:E-X, #11-A:X-P)
  • Electrophoresis (#3:S-P, #20:S-P, #11:E-S)
  • Gel extraction (#3:S-P, #20:S-P, #11:E-S, E-S vector, hycAp:E-S)

July 28th

  • Digestion(#1,3,5,11,hycAp)
  • Gel extraction(#1,3,5,11,hycAp)
  • Cloning(fhlA)


10th week: 7/29-8/4

July 30th

  • Digestion (#3:S-P, #11:X-P)
  • Electrophoresis (#3, #11)
  • Gel extraction (#3, #11)
  • Ligation (#3, #11)
  • Transformation (ligation product; #3-11)

August 1st

  • Digestion (#3:S-P, #11:X-P)
  • Electrophoresis (#3, #11)
  • Gel extraction (#3, #11)
  • Ligation (#3, #11)
  • Transformation (ligation product; #3-11)
  • Miniprep (#3, #5, #11)

August 2nd

  • Colony PCR (#3-11)
  • Culture (#3-11)
  • Colony pick up, culture (red colony of #11)
  • Digestion (#3:S-P, #5:E-X)

August 3th

  • Gel extraction (#3:S-P, #5:E-X)
  • Making solution of Kanamycin (100mg/mL)
  • Culture, miniprep (#11)
  • Culture (#11)

August 4th

  • Plating, Miniprep (#11)


August

11th week: 8/5-8/11

August 6th

  • Culture, Colony PCR (#3-11)

August 7th

  • Colony PCR, culture (#3-11)
  • Miniprep, Digestion, Gel extraction (culture product; #3-11)

August 8th

  • Digestion (#5: E-X)
  • Gel extraction (#5)
  • Miniprep (#5)
  • Colony PCR (culture product; #3-11)

August 9th

  • Colony PCR (#3-11)
  • Culture, Miniprep (#11)
  • Digestion (#11:X-P)
  • Cloning (#6,#8)
  • Ligation
  • Gel extraction (ligation product; #5, #11)
  • Ligation, Transformation (#3, #11)

August 10th

  • Colony PCR (#3-11)
  • Miniprep (#3)
  • Digestion (#3:S-P)
  • Cloning, Colony PCR (#6,#8)
  • cut check, PCR check (#3-11)
  • Electrophoresis (ligation product; #3, #11, PCR product; #6)
  • Gel extraction (#6)

August 11th

  • electrophoresis(PCR product; #3-11)
  • Digestion (#6:E-S)
  • electrophoresis (#8)


12th week: 8/12-8/18


August 13th

  • Check, Culture (#3-11)
  • Transformation (test plasmid)

August 15th

  • Gel extraction (cut check product; #3, #11)
  • Ligation, Transformation (#3-#11)
  • Make Vector for #6
  • Digestion (#3: E-S)
  • Gel extraction (digestion product; #3, #6(8/11)
  • PCR check (ligation product; #3-11)
  • Cloning (#6)

August 16th

  • Gel extraction (#6)
  • cloning (#6)
  • PCR check (#3-11)
  • PCR-cleanup (#8)
  • culture (#3-11)

August 17th

  • electrophoresis (#6)
  • Miniprep (#3-11)
  • Digestion (#3-#11: E-S, #6: E-S)
  • Gel extraction (#3-11)
  • Ligation, Transformation (#3-11-5)
  • Making solution of Ampicillin


13th week: 8/19-8/25


August 20th

  • PCR check (#3-11-5)
  • Digestion (#8: E-S)
  • Cloning (#6)
  • Digeston (#8: E-S)
  • Gel extraction (#6)

August 21th

  • Colony PCR (#1)
  • Digestion (#6: E-S, #3-11: E-S, #20: S-P)
  • Ligation (#3-11, #5)
  • Gel extraction (#3-11, #6)
  • Ligation (#6-pSB1A2)
  • Electrophresis (#3, #5; made previously)

August 22th

  • Cloning (#6; from test plasmid)
  • Colony PCR (#3-11-5(colony is not red), #20, 8/21 ligation product;#6, #8)
  • Miniprep,Frozen stock (culture product; #1, #5, #20)
  • Transformation (#8, #3-11-5)
  • Digestion (#6:X-S)
  • Making solution of Ampicillin
  • cloning retry (#6)

August 23th

  • PCR check (ligation product; #6)
  • Cloning (#6)

Colony PCR (#1, #6, #8, #11, #3-11-5, #3-11)

August 24th

  • Digestion (#6, E-S, #3-11, E-S)
  • Colony PCR #3-11
  • Electrophresis, Gel extraction (#6)
  • Vector insert (#6)
  • Sequencing (#8)

August 25th

  • Digestion (#6: E-S)
  • Colony PCR (#6-Vector)
  • Gel extraction (#3-11)


14th week: 8/26-9/2


August 26th

  • Electrophresis (#6)
  • Master plate (#6)
  • Colony PCR (#3-11-5)
  • Preculture (#6)

August 27th

  • Ligation (Miniprep product; #3-11, #5)
  • Digestion retry (#3-11, #5)
  • Miniprep (#6)
  • Mutagenesis (#6)
  • Sequencing (#6)

August 28th

  • Mutagenesis(#6)
  • Digestion(#3-11,#5)

August 29th

  • Mutagenesis by inverse PCR(#6)
  • Digestion(#5)
  • Cut check(#6)
  • PCR check(#6)
  • Sequencing(#6)
  • Making agar medium plate contain Chloramphenicol

August 30th

  • Digestion(#5,#3-11,#8)
  • Sequencing(#6)
  • Mutagenesis(#6)

August 31st

  • Colony PCR(#6)
  • Digestion(#3-11,#5)
  • Ligation(#3-11+#5)
  • Transformation(#3-11-5,#66)
  • Culture(#66)

September 1st

  • Cloning(#6)
  • Miniprep(#66)

September

15th week: 9/3-9/9


September 3rd

  • Miniprep(#3,#11)
  • Colony PCR(#3-11-5)
  • Transformation(#2)

September 4th

  • Digestion(#3,#6,#11)
  • Ligation(#3,#6)
  • Miniprep(#2)

September 5th

  • Ligation(#6+1A2)
  • Check PCR(#6)
  • Digestion(#2,8)

September 6th

  • Mutagenesis(#7)
  • Sequencing(#6)
  • Digestion(#5,#66)
  • Transformation(#2,#6)

September 7th

  • Colony PCR(#2,#8)
  • Cut check(EX,ES,XP,SPcut)
  • Digestion(#5,#66)
  • Ligation(#66+#5)
  • Transformation(#66-5)
  • Mutagenesis(#7)

September 8th

  • Sequencing(#6)
  • Colony PCR(#66-5)
  • Miniprep(#2)

16th week: 9/10-9/16


September 10th

  • Mutagenesis(#7)
  • Digestion(#2)
  • Transformation(#9,#40)

September 11th

  • Transformation(#44)
  • Mutagenesis(#7)
  • Miniprep(#6)
  • Colony PCR(#7)

September 12th

  • Culture(#44)
  • Miniprep(#44)
  • Mutagenesis(#7)
  • Transformation(#7)

September 13th

  • Culture(#1,#7,#20)
  • Colony PCR(#7)
  • Mutagenesis(#7)

September 14th

  • Inverse PCR(#10)
  • Digestion(#3,#7)
  • Ligation(#3-7)
  • Transformation(#3-7,#10)

September 15th

  • Digestion(#1,#7,#8,#44,#66)
  • Sequencing(#7,#10)
  • Colony PCR(#44,#3-7)

September 16th

  • Digestion(#1,#3,#7-5,#8,#10,#44,#66)
  • Transformation(#3-7-5,#3-10)


17th week: 9/17-9/23

September 17th

  • Miniprep(#7-5,#44)
  • Digetion(#3,5,10)
  • Transformation(#3-7-5,3-10-5)

September 18th

  • Miniprep(#7-5,#44)
  • Digetion(#3,#5,#10)
  • Transformation(#3-7-5,#3-10-5)

September 19th

  • Transformation(#4)
  • Miniprep(#3-7-5,#3-10-5)
  • Digestion(#3-7-5,#3-10-5,#20,#66)

September 20th

  • Miniprep(#4)
  • Digestion(#3-7-5,#3-10-5,#4,#20,#66)
  • Transformation(#20-3-7-5,#20-3-10-5,#4-4)

September 21st

  • Digestion(#3-7-5,#3-10-5,#4,#20,#66)
  • Transformation(#20-3-7-5,#20-3-10-5,#4-4,#7+1C3,#10+1C3)
  • Gas chromatography (preliminary experiment using WT)

September 22nd

  • Digestion(#3-7-5,#3-10-5,#4,#20,#66)
  • Transformation(#20-3-7-5,#20-3-10-5,#4-4)
  • Miniprep(#7+1C3,#10+1C3)

September 23rd

  • Digestion(#3-7-5,#3-10-5,#4,#8,#20,#66)
  • Transformation(#20-3-7-5,#20-3-10-5,#4-4)
  • Colony PCR(#20-3-7-5)

18th week: 9/24-9/26


September 24th

  • Digestion(#3-7-5,#3-10-5,#4,#8,#20,#66)
  • Transformation(#20-3-7-5,#20-3-10-5,#4-4)
  • Colony PCR(#20-3-7-5,#4+1C3)

September 25th

  • Digestion(#3-7-5,#3-10-5,#4,#20,#66)
  • Transformation(#20-3-7-5,#20-3-10-5,#4-4)
  • Colony PCR(#8-44)

Gas chromatography (WT,#6,#10)

September 26th

  • Digestion(#20-3-7-5,#20-3-10-5,#4,#66,#8-44)
  • Transformation(#8-44-20-3-7-5-1C3,#8-44-20-3-10-5-1C3)
  • Culture(4-4-1C3)
  • Gas chromatography (preparation of analytical line,WT,#7,#10)


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