Team:Biwako Nagahama/Original 3A Assembly

Contents 1 3A Assembly Biwako-Nagahama original protocol 2 Reason 3 “Protocol” 3.1 <iGEM Backbone(pSB1C3,pSB1K3,pSB11A3,pSB1T3) manufacture> 3.2 <Electrophoresis> 3.3 <Exo Star process> 3.4 <Phenol-chloroform extraction> 3.5 <EtOH precipitation> 3.6 <Electrophoresis> 3.7 <Restrictive Digestion> 3.8 <Phenol-chloroform extraction> 3.9 <EtOH precipitation> 3.10 <Restrictive Digestion> 3.11 <Phenol-chloroform extraction> 3.12 <EtOH precipitation> 3.13 <Electrophoresis> 3.14 <Ligation> 3.15 <Transformation> 3.16 <Colony PCR>(check Trance formation) 3.17 <Electrophoresis> 3.18 <Miniprep>

3A Assembly Biwako-Nagahama original protocol

Reason

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We succeeded in 3A Assembly using Linear Backbone from the Distribution Kit obtained from the iGEM Headquarter. But we could not succeeded in making 3A Assembly of the linear backbone taking reference of the Protocol of linear backbone found in the iGEM Homepage. So, we made our own 3A Assembly protocol suitable to our own lab environment that could increase the success rate of the experiment.

On discussing about the 3A Assembly with other participating teams, we found that other teams were not preparing their own protocol regarding 3A Assembly. We found that the 3A Assembly could be performed easily in the environment with certain restrictions. Because 3A Assembly is Assmbly method of the high probability not to need PCR and gel purification. So we tried to debug the available protocols and make our own protocols suitable to our own experimenting environment and help other teams with similar environment condition.

“Protocol”

<iGEM Backbone(pSB1C3,pSB1K3,pSB11A3,pSB1T3) manufacture>

2×KODFx buffer・・・25μL

dNTPs[2mM]・・・0μL

[http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones ※1] SB-prep-3P-1・・・1.5μL

[http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones ※2] SB-prep-2Ea・・・1.5μL

KODFx[1.0U/μL]・・・1.0μL

MilliQ H2O・・・10.0μL

Template[1～10ng/μL](pSB1C3 or pSB1K3 or pSB1T3 or pSB1A3)・・・1.0μL

Total・・・50.0μL

[http://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_002.html ToYoBo KOD FX]<p/> <p>↓

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94℃　2min

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98℃　10sec

58℃   30sec             30cycles

68℃   2min30sec

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10℃　∞

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<Electrophoresis>

TE buffer・・・7μL

PCR Sample・・・2μL

10×Loading buffer・・・1μL

Total・・・10μL

0.7% Gel

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1 λ-HindⅢ            10μL

2 500bp DNA ladder  10μL

3 pSB1A3                10μL

4 pSB1K3                10μL

5 pSB1T3                10μL

6 pSB1C3                10μL

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<Exo Star process>

ExoⅠ・・・0.1μL

BAP・・・0.1μL

MilliQ H2O・・・1.8μL

PCR Sample・・・17.0μL

Total・・・19.0μL

[http://193.218.17.133/ex/downloads/brochures/life_science/ge_illustra_exostar.pdf illustra™ ExoStar™ 1-Step]

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37℃ 30min

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80℃ 15min

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10℃ ∞

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<Phenol-chloroform extraction>

Add Phenol-chloroform where is equivalent to Exo Star process sample

↓Centrifuge 4℃ 13,000rpm 5min

Only supernatant was taken

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<EtOH precipitation>

Add 1μL 20mg/mL Glycogen

↓Mix

Add 1/10 volume 3M CH3COONa(pH5.2)

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Add 2.5 times volume 99.5% EtOH

↓Vortex

↓Centrifuge 4℃ 13,000rpm 20min

Waste supernatant

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Add 500μL 70% EtOH

↓Mix

↓Centrifuge for 10s in a table-top microcentrifuge

Waste supernatant

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65℃ Dry up

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Add 11μL TE buffer

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<Electrophoresis>

TE buffer・・・8μL

EtOH crystallization Sample・・・1μL

10×Loading buffer・・・1μL

Total・・・10μL

0.7% Gel

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<Restrictive Digestion>

Backbone template・・・500ng

dH2O・・・-50μL

10×H buffer・・・5μL

EcoRⅠ・・・1μL

PstⅠ・・・1μL

Total・・・50μL

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37℃ 1h

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<Phenol-chloroform extraction>

Add Phenol-chloroform where is equivalent to Exo Star process sample

↓Centrifuge 4℃ 13,000rpm 5min

Only supernatant was taken

↓

<EtOH precipitation>

Add 1μL 20mg/mL Glycogen<p> <p>↓Mix

Add 1/10 volume 3M CH3COONa(pH5.2)

↓

Add 2.5 times volume 99.5% EtOH

↓Vortex

↓Centrifuge 4℃ 13,000rpm 20min

Waste supernatant

↓

Add 500μL 70% EtOH

↓Mix

↓Centrifuge for 10s in a table-top microcentrifuge

Waste supernatant

↓

65℃ Dry up

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Add 10μL TE buffer

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<Restrictive Digestion>

EtOH precipitation Sample・・・10μL

dH2O・・・33μL

10×H buffer・・・5μL

EcoRⅠ・・・1μL

PstⅠ・・・1μL

Total・・・50μL

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37℃ Over Night

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<Phenol-chloroform extraction>

Add Phenol-chloroform where is equivalent to Exo Star process sample

↓Centrifuge 4℃ 13,000rpm 5min

Only supernatant was taken

↓

<EtOH precipitation>

Add 1μL 20mg/mL Glycogen

↓Mix

Add 1/10 volume 3M CH3COONa(pH5.2)

↓

Add 2.5 times volume 99.5% EtOH

↓Vortex

↓Centrifuge 4℃ 13,000rpm 20min

Waste supernatant

↓

Add 500μL 70% EtOH

↓Mix

↓Centrifuge for 10s in a table-top microcentrifuge

Waste supernatant

↓

65℃ Dry up

↓

Add 8μL TE buffer

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<Electrophoresis>

TE buffer・・・8μL

EtOH crystallization Sample・・・1μL

10×Loading buffer・・・1μL

Total・・・10μL

0.7% Gel

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<Ligation>

①insertA(20ng)

②insertB(20ng)

③Backbone template(20ng)

DNA Ligation Kit ver 2.1　　①+②+③μL

[http://www.clontech.com/takara/JP/Products/Molecular_Biology/Cloning_Mapping_and_cDNA_Synthesis/Quick_DNA_Ligation?sitex=10036:22372:US TaKaRa DNA Ligation Kit ver 2.1]

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16℃ 30min

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<Transformation>

Add all ligation solution into your desired 100μL of competent cells

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Hold on ice for 20min

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Heat shock at 42℃ for 30sec

↓quickly

On ice for 2min

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Add 900μL of SOCborth

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Hold at 37℃ for 30min

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Plating 100μL of DNA Transformation

↓Centrifuge 4℃ 13,000rpm 1min

Waste supernatant for 800μL, and pipetting on anther plate

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Plating all

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Incubate at 37℃ (over night)

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<Colony PCR>(check Trance formation)

Take a single colony from the plate and then mix it with 30μL of de-ionized water(dH2O)

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dH2O・・・12.7μL

10×NH4 buffer・・・2.5μL

50mM MgCl2・・・0.75μL

25mM dNTPs・・・2μL

10μM [http://parts.igem.org/Part:BBa_G1004 ※3] BBa_G1004・・・1μL

10μM [http://parts.igem.org/Part:BBa_G1005 ※4] BBa_G1005・・・1μL

BIO Taq(5U/μL)・・・0.05μL

Template(dH2O mixed colony)・・・5μL

Total・・・25μL

BIOTAQ™ DNA Polymerase

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95℃　30sec

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95℃　10sec

55℃ 20sec 30cycles

72℃ 2min

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72℃　2min

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10℃　∞

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<Electrophoresis>

PCR Sample・・・9μL

10×Loading buffer・・・1μL

Total・・・10μL

0.7% Gel

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1 500bp DNA ladder      10μL

2～16 Sample1～15     10μL

17 500bp DNA ladder     10μL

18 500bp DNA ladder     10μL

19～29 Sample16～30   10μL

30 500bp DNA ladder     10μL

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Sample1～Sample30:T7RNApolymerase-Double Terminater on pSB1K3

<Miniprep>

1.5mL Culture fluid→New 1.5mL tube

↓Centrifuge 4℃ 13,000rpm 1min

Waste supernatant

↓

Add 200μl SolutionⅠ

↓vortex

Add 200μl SolutionⅡ

↓Mix(softy)

Add 200μl SolutionⅢ

↓Mix(softy)

Check to become cloudy

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On ice 5min

↓Centrifuge 4℃ 13,000rpm 10min

supernatant→New 1.5mL tube

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Add 500μL Isopropyl alchol

↓vortex

↓Centrifuge 4℃ 13,000rpm 20min

Waste supernatant

↓

Add 500μL 70% EtOH

↓Mix

↓Flash

Waste supernatant

↓

65℃ Dry up

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Add 50μL RNase in TE buffer

↓Mix

37℃ 20min

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1 λ-HindⅢ ladder      10μL

2 500bp DNA ladder     10μL

3 T7RNApolymerase-duble.terminater     10μL

4 T7RNApolymerase-duble.terminater     10μL

5 T7RNApolymerase-duble.terminater  10μL

6 T7RNApolymerase-duble.terminater     10μL

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By. Syohei Takeshita