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Team:Chiba/Notebook 2
From 2010.igem.org
2010-09-21
PCRBecause it didn’t come out vector PCR(gel electrophoresis), we operated re-experiment to change template and template amounts.
Using template 1. Biobrick 2010 1-3A(pSB1C3) 2. Biobrick 2010 pSB1C3 3. Biobrick 2010 2-9A(pSB1A3) 4. control
Using primer Fwd : pSB1C3 FASTR-F Rev : pSB1C3 FASTR-R
------------------------- template 5㎕ primer Fwd 5㎕ primer Rev 5㎕ dNTP 5㎕ 10xbuffer 5㎕ NFW 24㎕ VentP 1㎕ ------------------------- 50㎕
PCR(TAKARA) 94°C – 5min 94°C – 15sec -- 51°C – 30sec │- 25 cycles 72°C – 1min -- 72°C – 10min
Vector PCR
Using template 1. Biobrick 2010 1-3A(pSB1C3) 2. Biobrick 2010 pSB1C3 3. Biobrick 2010 2-9A(pSB1A3) 4. control
------------------------- template 3㎕ primer Fwd 5㎕ primer Rev 5㎕ dNTP 5㎕ 10xbuffer 5㎕ NFW 26㎕ VentP 1㎕ ------------------------- 50㎕
Using primer Fwd : pSB1C3 FASTR-F Rev : pSB1C3 FASTR-R
PCR Condition 94°C – 5min 94°C – 30sec -- 51°C – 30sec │-25 cycles 72°C – 2min -- 72°C – 10min
2010-09-22
Biobrick 2010 2-9A(pSB1A3) -> Gel extraction Gel ElectrophoresisBiobrick 2010 2-9A(pSB1A3), Biobrick 2010 1-3A(pSB1C3), Biobrick 2010 pSB1C3, Biobrick 2010 2-9A(pSB1A3) As a result, it didn’t come out band of 1-3A and pSB1C3. We concluded re-experiment from liquid incubation to mini-prep. Must check gel electrophoresis band after mini-prep.
FASTR Cloning (Plasmid1)
Vector (about 2kbp) -pSB1A3 (conservative solution 100ng/㎕)
Insert (about 1kbp) 1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕) 2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕) 3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕) 4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
master mix --------------------------------------------------------------------- Vector(50ng) 0.5 2 Insert(75ng) 3 - Tango buffer 0.5 2 T4 Ligase buffer 0.5 2 ATP 1 4 LguI 0.5 2 Ligase 0.5 2 DpnI 0.5 2 NFW 3 12 ----------------------------------------------------------------------- Total 10㎕ 28㎕ Room temperature 2h
Transformation
Using cell strain : BL21(DE3) 50㎕ Using plasmid L1 : Pet-LuxR-PT7/cI(OR2)-GFP 5㎕ L2 : Pet-LuxR-PT7/cI(OR1)-GFP 5㎕ L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕ L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕
After transformation
We added IPTG(0μM / 100μM / 200μM) to each cell strain. It became 12 plates. ※A Reason of using DE3 cell strain DE3 cell strain has T7 RNA Polymerase gene in genome, and under IPTG derivation T7RNAP is expressed. Plasmid1 has T7/cI hybrid promoter. So if this promoter is accelerated by T7RNAP, GFP is expressed and shines green. In other words, we used DE3 cell strain to check T7 accelerating function of PT7/cI.
2010-09-23
From yesterday, we did mini-prep liquid incubated sample 1-3A and pSB1C3. And then it is eluted by 50㎕ Elution Solution. ↓ Gel Electrophoresis(1㎕)L1 : Pet-LuxR-PT7/cI(OR2)-GFP 5㎕ L2 : Pet-LuxR-PT7/cI(OR1)-GFP 5㎕ L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕ L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕ Remaining of above sample is transformed and incubated. cell strain : XL10G(50㎕/tube) plasmid : L1~L4(total 4)
FASTR Cloning (Plasmid1)
Vector (about 2kbp) -pSB1A3 (conservative solution 100ng/㎕) Insert (about 1kbp) 1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕) 2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕) 3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕) 4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕) master mix --------------------------------------------------------------------- Vector(50ng) 0.5 2 Insert(75ng) 3 - Tango buffer 0.5 2 T4 Ligase buffer 0.5 2 ATP 1 4 LguI 0.5 2 Ligase 0.5 2 DpnI 0.5 2 NFW 3 12 ----------------------------------------------------------------------- Total 10㎕ 28㎕ Room temperature 2h
After ligation plasmid is transformed and incubated in LB broth. cell strain : BL21(DE3), XL10G(each 50㎕/tube) plasmid : L1~L4
2010-09-24
At 23th September Remaining of above sample is transformed and incubated. cell strain : XL10G(50㎕/tube) plasmid : L1~L4(total 4) We picked 4 colonies of each plate out from L1~L4 plates and operated colony PCR. Moreover, we did liquid incubation each colony.Insert Check Colony PCR(Plasmid 1)
master mix -------------------------------------------------------------------------- Template(picked colonies) Primer VF 2㎕ 40㎕ Primer VR 2㎕ 40㎕ 10x Buffer 2㎕ 40㎕ dNTP 2㎕ 40㎕ NFW 10㎕ 200㎕ Taq DNA Polymerase 0.5㎕ 5㎕ -------------------------------------------------------------------------- Total 20㎕ 400㎕ ↓ PCR 94°C – 5min 94°C – 30sec -- 51°C – 30sec │-20 cycles 72°C – 1min -- 72°C – 10min ↓ Gel Electrophoresis
2010-09-25
Mini-prep and gel electrophoresis of liquid culture medium at 24th September.Gel Electrophoresis picture ↓ Transformation to BL21(DE3) ↓ Incubation in LB broth, IPTG(0, 10, 100 μM) ↓37°C About total sample, it is shown GFP ON/OFF switching on eyes. ↓ For the purpose of measuring fluorescence level, we did liquid incubation from each plate. IPTG(0, 10, 100 μM) x 6 samples = 18 test tubes ↓37°C ∙ measuring fluorescence level(liquid incubation) ∙ pelletization of cell
Digestion and ligation of BBa-J01011(Ptet-cI) and pSB3C5
(Digestion)
Insert(J01011) Vector(pSB3C5)
DNA 10㎕ 10㎕
NFW 32.5㎕ 32.5㎕
NEB Buffer 2 5㎕ 5㎕
BSA 0.5㎕ 0.5㎕
EcoRI 1㎕ 1㎕
PstI 1㎕ 1㎕
------------------------------------------------------------------------
total 50㎕ 50㎕
↓37°C 15min incubator
↓65°C 20min (sterilization dryer)
※After digestion, we did gel electrophoresis
(Ligation)
13㎕ NFW
2㎕ Insert(digested)
2㎕ Vector(digested) total 20㎕ in 0.2mL PCR tube
2㎕ 10x T4 DNA Ligase Buffer
1㎕ T4 DNA Ligase
↓10min at room temperature
↓20min at 65°C (sterilization dryer)
(Transformation)
Transformation XL10G 50㎕ to 10㎕ of ligation product
↓37°C
Insert check of living colonies(colony PCR) / liquid incubation
↓total 7 colonies ↓37°C
Insert Check Colony PCR(Plasmid 1) ↓We did mini-prep only No.4 sample checked insert.
--------------------------------------------
Template(picked colonies) ↓
Primer VF 2㎕ Gel electrophoresis
Primer VR 2㎕
10x Buffer 2㎕
dNTP 2㎕
NFW 11.5㎕
Taq DNA Polymerase 0.5㎕
--------------------------------------------------------
Total 20㎕
↓
PCR
94°C 5min
94°C 30sec --
51°C 30sec │-30 cycle
72°C 1min --
72°C 10min
↓
Gel electrophoresis
