Team:KAIT Japan/Notebook

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__NOTOC__
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='''Notebook'''=
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:We made an experiment every day. It was process of trial and error.
 +
=='''Creating parts of Methylation and Azurin'''==
 +
:<big>'''Date:8/9'''</big> PCR
-
We are creating now...<br>
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:<big>'''Date:8/10'''</big> Electrophoresis
-
=Creating parts of Tar methylation region.=
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-
==Date:8/9==
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-
===Colony PCR===
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-
Reagent
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-
:*TaKaRa Ex Taq(5units/μL) 0.5μL
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-
:*10×Ex Taq buffer 10μL
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-
:*dNTP Mixture(2.5Meach) 8μL
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-
:*Primer F(10μM) 4μL
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-
:*Primer R(10μM) 4μL
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-
:*Template(E.coli DH5α)
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::'''→Reflection:We Took E.coli too many.You should take less E.coli.'''<br>
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-
Conditions of the thermal cycler
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-
#95°C(5min)
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-
#94°C(30sec)
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-
#61°C(30sec)
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-
#71°C(40sec)
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-
#72°C(1min)
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#4°C(Save)
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#*2-4:35cycle'''→Reflection:The number of cycles was less.So,We increased The number of cycles in 8/11.(50 cycles) '''
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#*gradient:60-61°C(+0.1°C)
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-
----
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:<big>'''Date:8/13'''</big> Electrophoresis and The refinement of DNA,PCR
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==Date:8/11==
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:<big>'''Date:8/14'''</big> Transformation
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===The purified DNA===
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#Electrophoresis
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#*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL
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#*Sample:dye 1μL,sample 5μL
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#*Gel concentration:1.2%,Migration time:30min
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-
:::'''→Reflection:Band was less.'''
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#Storage
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===PCR Product===
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:<big>'''Date:8/16'''</big> Blue white selection and Miniprep,Colony PCR
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#Electrophoresis
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#*The gel check and cut
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#DNA purification
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#Confirmation of electrophoresis
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#PCR
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#*50cycle
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#Storage
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-
----
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:<big>'''Date:8/17'''</big> Electrophoresis and Colony PCR
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==Date:8/13==
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:<big>'''Date:8/18'''</big> PCR and Electrophoresis
-
===Confirmed of electrophoresis by PCR product and Ligation of the TA vector===
+
-
#Electrophoresis
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-
#*Gel concentration:1.2%,Migration time:30min
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-
#*Marker:Flash Gel 5μL
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-
#*Sample:dye 1μL,sample 5μL
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-
#Check and Colony PCR
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-
#Add to TA vector
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#*PCR product 2μL
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#*pMD20-Tvector 1μL
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#*D<sub>2</sub>W 2μL
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#*Ligation Mighty Mix 5μL
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#Heat insulation(16°C,30min)
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#Storage(-20°C)
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-
----
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:<big>'''Date:8/20'''</big> Extraction and the refinement of DNA
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==Date:8/14==
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:<big>'''Date:8/21'''</big> PCR and Electrophoresis,Transformation
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===Transformation===
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#Put competent cells on ice(10-15min)
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#Add Ligation reaction solution(10μL) and tapping
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#On the ice(30min)[Transformation]
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#Add LB medium(0.7mL)
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#Incubate(60min,37°C)
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#Add X-gal(40μL) and ampicillin(10μL)[200μg/mL] on LB agar medium(IPTG)
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#Add one incubated(100μL)
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#Cultivation(overnight)
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-
----
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:<big>'''Date:8/22'''</big> Electrophoresis
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==Date:8/18==
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:<big>'''Date:8/23'''</big> PCR
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===Colony PCR===
+
-
Reagent
+
-
:*TaKaRa Ex Taq(5units/μL) 0.5μL
+
-
:*10×Ex Taq buffer 10μL
+
-
:*dNTP Mixture(2.5Meach) 8μL
+
-
:*Primer F(10μM) 4μL
+
-
:*Primer R(10μM) 4μL
+
-
:*Template(E.coli DH5α)
+
-
:*sterilized water(73.5μL)
+
-
Conditions of the thermal cycler
+
-
#95°C(5min)
+
-
#94°C(30sec)
+
-
#61°C(30sec)
+
-
#71°C(40sec)
+
-
#72°C(1min)
+
-
#4°C(Save)
+
-
#*2-4:30cycle
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-
#*gradient:57-62°C(+0.1c)
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-
----
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:<big>'''Date:8/25'''</big> Miniprep and Restriction enzyme processing
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==Date:8/20==
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:<big>'''Date:8/27'''</big> Electrophoresis
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===DNA extraction and purification of P.aeruginosa===
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#Centrifuge culture medium(6,000rpm,5min,4°C)
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:<big>'''Date:8/28'''</big> Electrophoresis and The refinement of DNA,PCR
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#Remove supernatant,Add saline[0.85%](1.5mL)
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#Centrifuge(6,000rpm,5min,4°C)
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:<big>'''Date:8/29'''</big> Electrophoresis
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#Add 5mMEDTA 1mL
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#Add 10%SDS 100μL
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:<big>'''Date:8/30'''</big> Electrophoresis and The refinement of DNA
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Add proteinase K 50μL
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#Vortex
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:<big>'''Date:9/1'''</big> Extraction of DNA
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#Incubation(30min,55°C)
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#Add phenol mixture(TE saturated phenol:chloroform:isoamyl alcohol=25:24:1)
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:<big>'''Date:9/2'''</big> Redesign of the primer
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#Shake vigorously(1min)
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#*At this time,It became muddy white in color.
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:<big>'''Date:9/3'''</big> PCR and Restriction enzyme processing,Electrophoresis
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#Centrifuge(16,000rpm,10min,4°C)
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#Pick up supernatant,remove new microtube
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:<big>'''Date:9/5'''</big> Miniprep
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#Repeat step 7-11
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#Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
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:<big>'''Date:9/6'''</big> Electrophoresis
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#Vortex
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#Wind the DNA by a thin glass rod.
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:<big>'''Date:9/10'''</big> Colony PCR
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#Rinse chilled 70%-ethanol(500μL)
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#Pick up DNA,air dry
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:<big>'''Date:9/12'''</big> Miniprep
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#Add TE buffer 500μL
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#Add RNase A 50μL
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:<big>'''Date:9/14'''</big> PCR
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#Incubation(20min,37°C)
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-
#Add proteinase K 50μL
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:<big>'''Date:9/15'''</big> Restriction enzyme processing
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#Incubation(1h,37°C)
+
 
-
#Add phenol mixture
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:<big>'''Date:9/16'''</big> PCR
-
#Vortex(1min)
+
 
-
#Centrifuge(16,000rpm,10min,4°C)
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:<big>'''Date:9/17'''</big> PCR
-
#Pick up supernatant,remove new microtube
+
 
-
#Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
+
:<big>'''Date:9/19'''</big> PCR and Restriction enzyme processing
-
#Wind the DNA by a thin glass rod.
+
 
-
#Rinse chilled 70%-ethanol(500μL,about 30s)
+
:<big>'''Date:9/20'''</big> Ligation
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#Pick up DNA,air dry
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-
#Add TE buffer 200μL
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:<big>'''Date:9/22'''</big> Transformation
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#*Melt DNA in buffer
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 +
:<big>'''Date:9/23'''</big> PCR and Electrophoresis
 +
 
 +
:<big>'''Date:9/24'''</big> Ligation
 +
 
 +
:<big>'''Date:9/25'''</big> Transformation
 +
 
 +
:<big>'''Date:9/27'''</big> Miniprep and Electrophoresis
 +
 
 +
=='''Creating parts of Tar'''==
 +
:<big>'''Date:9/13'''</big> PCR and Colony PCR
 +
 
 +
:<big>'''Date:9/14'''</big> Electrophoretic confirmation
 +
 
 +
:<big>'''Date:9/15'''</big> Restriction enzyme processing
 +
 
 +
:<big>'''Date:9/16'''</big> Electrophoresis of the restriction enzyme processing product and Restriction enzyme processing
 +
 
 +
:<big>'''Date:9/17'''</big> Refinement of DNA and Ligation,Transformation
 +
 
 +
:<big>'''Date:9/18'''</big> Making of a master plate and Preculture,Recultivation
 +
 
 +
:<big>'''Date:9/19'''</big> Colony PCR and Electrophoresis
 +
 
 +
:<big>'''Date:9/20'''</big> Ligation and Transformation
 +
 
 +
:<big>'''Date:9/21'''</big> Inoculation
 +
 
 +
:<big>'''Date:9/22'''</big> Making of a master plate and Colony PCR,Electrophoresis,Preculture
 +
 
 +
:<big>'''Date:9/23'''</big> Electrophoretic confirmation and Electrophoresis of the PCR
 +
 
 +
:<big>'''Date:9/24'''</big> Miniprep and Electrophoresis
 +
 
 +
:<big>'''Date:9/25'''</big> PCR of the miniprep product and Electrophoresis
 +
 
 +
=='''Creating parts of EnvZ'''==
 +
:<big>'''Date:9/10'''</big> Design of the primer and PCR,Elecrtophoresis,Miniprep
 +
 
 +
:<big>'''Date:9/13'''</big> PCR and Elecrtophoresis
 +
 
 +
:<big>'''Date:9/18'''</big> Design of the primer
 +
 
 +
:<big>'''Date:9/19'''</big> PCR and Elecrtophoresis
 +
 
 +
:<big>'''Date:9/20'''</big> Restriction enzyme processing
 +
 
 +
:<big>'''Date:9/21'''</big> Ligation and PCR,Elecrtophoresis
 +
 
 +
:<big>'''Date:9/23'''</big> PCR
 +
 
 +
:<big>'''Date:9/24'''</big> The refinement of DNA and Restriction enzyme processing,Ligation
 +
 
 +
:<big>'''Date:9/25'''</big> Transformation
 +
 
 +
:<big>'''Date:9/26'''</big> Liquid culture
 +
 
 +
|}

Latest revision as of 22:12, 26 September 2012

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Home

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Project

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Parts

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Protocol

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Notebook

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Results

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Safety

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Human Practice

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Team

Notebook

We made an experiment every day. It was process of trial and error.

Creating parts of Methylation and Azurin

Date:8/9 PCR
Date:8/10 Electrophoresis
Date:8/13 Electrophoresis and The refinement of DNA,PCR
Date:8/14 Transformation
Date:8/16 Blue white selection and Miniprep,Colony PCR
Date:8/17 Electrophoresis and Colony PCR
Date:8/18 PCR and Electrophoresis
Date:8/20 Extraction and the refinement of DNA
Date:8/21 PCR and Electrophoresis,Transformation
Date:8/22 Electrophoresis
Date:8/23 PCR
Date:8/25 Miniprep and Restriction enzyme processing
Date:8/27 Electrophoresis
Date:8/28 Electrophoresis and The refinement of DNA,PCR
Date:8/29 Electrophoresis
Date:8/30 Electrophoresis and The refinement of DNA
Date:9/1 Extraction of DNA
Date:9/2 Redesign of the primer
Date:9/3 PCR and Restriction enzyme processing,Electrophoresis
Date:9/5 Miniprep
Date:9/6 Electrophoresis
Date:9/10 Colony PCR
Date:9/12 Miniprep
Date:9/14 PCR
Date:9/15 Restriction enzyme processing
Date:9/16 PCR
Date:9/17 PCR
Date:9/19 PCR and Restriction enzyme processing
Date:9/20 Ligation
Date:9/22 Transformation
Date:9/23 PCR and Electrophoresis
Date:9/24 Ligation
Date:9/25 Transformation
Date:9/27 Miniprep and Electrophoresis

Creating parts of Tar

Date:9/13 PCR and Colony PCR
Date:9/14 Electrophoretic confirmation
Date:9/15 Restriction enzyme processing
Date:9/16 Electrophoresis of the restriction enzyme processing product and Restriction enzyme processing
Date:9/17 Refinement of DNA and Ligation,Transformation
Date:9/18 Making of a master plate and Preculture,Recultivation
Date:9/19 Colony PCR and Electrophoresis
Date:9/20 Ligation and Transformation
Date:9/21 Inoculation
Date:9/22 Making of a master plate and Colony PCR,Electrophoresis,Preculture
Date:9/23 Electrophoretic confirmation and Electrophoresis of the PCR
Date:9/24 Miniprep and Electrophoresis
Date:9/25 PCR of the miniprep product and Electrophoresis

Creating parts of EnvZ

Date:9/10 Design of the primer and PCR,Elecrtophoresis,Miniprep
Date:9/13 PCR and Elecrtophoresis
Date:9/18 Design of the primer
Date:9/19 PCR and Elecrtophoresis
Date:9/20 Restriction enzyme processing
Date:9/21 Ligation and PCR,Elecrtophoresis
Date:9/23 PCR
Date:9/24 The refinement of DNA and Restriction enzyme processing,Ligation
Date:9/25 Transformation
Date:9/26 Liquid culture
Retrieved from "http://2012.igem.org/Team:KAIT_Japan/Notebook"