Team:KAIT Japan/Notebook

From 2012.igem.org

(Difference between revisions)
(→Miniprep)
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==Date:8/9==
==Date:8/9==
===Colony PCR===
===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)
 
::'''→Reflection:We Took E.coli too many.You should take less E.coli.'''<br>
::'''→Reflection:We Took E.coli too many.You should take less E.coli.'''<br>
-
Conditions of the thermal cycler
+
::'''→Reflection:The number of cycles was less.So,We increased The number of cycles in 8/11.(50 cycles) '''
-
#95°C(5min)
+
-
#94°C(30sec)
+
-
#61°C(30sec)
+
-
#71°C(40sec)
+
-
#72°C(1min)
+
-
#4°C(Save)
+
-
#*2-4:35cycle'''→Reflection:The number of cycles was less.So,We increased The number of cycles in 8/11.(50 cycles) '''
+
-
#*gradient:60-61°C(+0.1°C)
+
----
----
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==Date:8/11==
==Date:8/11==
===The purified DNA===
===The purified DNA===
-
#Electrophoresis
 
-
#*Marker:dye 1&mu;L,DNA molecule 2&mu;L,TE buffer 3&mu;L
 
-
#*Sample:dye 1&mu;L,sample 5&mu;L
 
-
#*Gel concentration:1.2%,Migration time:30min
 
:::'''→Reflection:Band was less.'''
:::'''→Reflection:Band was less.'''
-
#Storage
 
===PCR Product===
===PCR Product===
-
#Electrophoresis
 
-
#*The gel check and cut
 
-
#DNA purification
 
-
#Confirmation of electrophoresis
 
-
#PCR
 
-
#*50cycle
 
-
#Storage
 
----
----
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==Date:8/13==
==Date:8/13==
===Confirmed of electrophoresis by PCR product and Ligation of the TA vector===
===Confirmed of electrophoresis by PCR product and Ligation of the TA vector===
-
#Electrophoresis
 
-
#*Gel concentration:1.2%,Migration time:30min
 
-
#*Marker:Flash Gel 5&mu;L
 
-
#*Sample:dye 1&mu;L,sample 5&mu;L
 
-
#Check and Colony PCR
 
-
#Add to TA vector
 
-
#*PCR product 2&mu;L
 
-
#*pMD20-Tvector 1&mu;L
 
-
#*D<sub>2</sub>W 2&mu;L
 
-
#*Ligation Mighty Mix 5&mu;L
 
-
#Heat insulation(16°C,30min)
 
-
#Storage(-20°C)
 
----
----
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==Date:8/14==
==Date:8/14==
===Transformation===
===Transformation===
-
#Put competent cells on ice(10-15min)
 
-
#Add Ligation reaction solution(10&mu;L) and tapping
 
-
#On the ice(30min)[Transformation]
 
-
#Add LB medium(0.7mL)
 
-
#Incubate(60min,37°C)
 
-
#Add X-gal(40&mu;L) and ampicillin(10&mu;L)[200&mu;g/mL] on LB agar medium(IPTG)
 
-
#Add one incubated(100&mu;L)
 
-
#Cultivation(overnight)
 
----
----
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==Date:8/25==
==Date:8/25==
===Miniprep===
===Miniprep===
-
#Add culture medium 1mL in a microtube.
 
-
#Centrifuge(1min,4°C,12,000rpm).
 
-
#Remove the supernatant.
 
-
#Repeat 1-3.
 
-
#Add SolI 100&mu;L and Vortex.
 
-
#Centrifuge(1min,4°C,12,000rpm).
 
-
#Add SolII 200&mu;L and invert.
 
-
#ice-cold 3min.
 
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#Add SolIII 150&mu;L and invert.
 
-
#ice-cold 5min.
 
-
#Centrifuge(5min,4°C,12,000rpm).
 
-
#
 
=Creating parts of azurin=
=Creating parts of azurin=
==Date:8/16==
==Date:8/16==
===Colony PCR===
===Colony PCR===
-
Reagent
 
-
:*TaKaRa Ex Taq(5units/&mu;L) 0.5&mu;L
 
-
:*10×Ex Taq buffer 10&mu;L
 
-
:*dNTP Mixture(2.5Meach) 8&mu;L
 
-
:*Primer F(10&mu;M) 4&mu;L
 
-
:*Primer R(10&mu;M) 4&mu;L
 
-
:*Template(E.coli DH5α)
 
-
:*sterilized water(73.5&mu;L)
 
-
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
 
-
#*gradient:57-62°C(+0.1c)
 
===Ligation===
===Ligation===
-
Reagent
+
 
-
:*sterilize water 2&mu;L
+
-
:*PCR product 2&mu;L
+
-
:*vector DNA 1&mu;L
+
-
:*Ligation Mighty Mix 5&mu;L
+
-
Method
+
-
#Incubation(1h,16°C)
+
-
#Storage Overnight(-4°C)
+
----
----
==Date:8/18==
==Date:8/18==
===Colony PCR===
===Colony PCR===
-
Reagent
 
-
:*TaKaRa Ex Taq(5units/&mu;L) 0.5&mu;L
 
-
:*10×Ex Taq buffer 10&mu;L
 
-
:*dNTP Mixture(2.5Meach) 8&mu;L
 
-
:*Primer F(10&mu;M) 4&mu;L
 
-
:*Primer R(10&mu;M) 4&mu;L
 
-
:*Template(E.coli DH5α)
 
-
:*sterilized water(73.5&mu;L)
 
-
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
 
-
#*gradient:57-62°C(+0.1c)
 
----
----
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==Date:8/20==
==Date:8/20==
===DNA extraction and purification of ''P.aeruginosa''===
===DNA extraction and purification of ''P.aeruginosa''===
-
#Centrifuge culture medium(6,000rpm,5min,4°C)
 
-
#Remove supernatant,Add saline[0.85%](1.5mL)
 
-
#Centrifuge(6,000rpm,5min,4°C)
 
-
#Add 5mMEDTA 1mL
 
-
#Add 10%SDS 100&mu;L
 
-
#Add proteinase K 50methodL
 
-
#Vortex
 
-
#Incubation(30min,55°C)
 
-
#Add phenol mixture(TE saturated phenol:chloroform:isoamyl alcohol=25:24:1)
 
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#Shake vigorously(1min)
 
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#*At this time,It became muddy white in color.
 
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#Centrifuge(16,000rpm,10min,4°C)
 
-
#Pick up supernatant,remove new microtube
 
-
#Repeat step 7-11
 
-
#Add 3M-sodium acetate 40&mu;L,chilled isopropanol 400&mu;L
 
-
#Vortex
 
-
#Wind the DNA by a thin glass rod.
 
-
#Rinse chilled 70%-ethanol(500&mu;L)
 
-
#Pick up DNA,air dry
 
-
#Add TE buffer 500&mu;L
 
-
#Add RNase A 50&mu;L
 
-
#Incubation(20min,37°C)
 
-
#Add proteinase K 50&mu;L
 
-
#Incubation(1h,37°C)
 
-
#Add phenol mixture
 
-
#Vortex(1min)
 
-
#Centrifuge(16,000rpm,10min,4°C)
 
-
#Pick up supernatant,remove new microtube
 
-
#Add 3M-sodium acetate 40&mu;L,chilled isopropanol 400&mu;L
 
-
#Wind the DNA by a thin glass rod.
 
-
#Rinse chilled 70%-ethanol(500&mu;L,about 30s)
 
-
#Pick up DNA,air dry
 
-
#Add TE buffer 200&mu;L
 
-
#*Melt DNA in buffer
 
|}
|}

Revision as of 16:37, 2 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

Contents

Creating parts of Tar methylation region

Date:8/9

Colony PCR

→Reflection:We Took E.coli too many.You should take less E.coli.
→Reflection:The number of cycles was less.So,We increased The number of cycles in 8/11.(50 cycles)

Date:8/11

The purified DNA

→Reflection:Band was less.

PCR Product

Date:8/13

Confirmed of electrophoresis by PCR product and Ligation of the TA vector

Date:8/14

Transformation

Date:8/25

Miniprep

Creating parts of azurin

Date:8/16

Colony PCR

Ligation

Date:8/18

Colony PCR

Date:8/20

DNA extraction and purification of P.aeruginosa

Retrieved from "http://2012.igem.org/Team:KAIT_Japan/Notebook"