From 2011.igem.org

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Revision as of 16:54, 22 September 2011

Lab Work

Week1
Week2
Week3
Week4
Week5
Week6
Week7
Week8
Week9
Protocol

Week1: Monday 1st - Sunday 7th August

Monday
行った実験名：

使った試薬名、容量：

用いた機械：

行った人：

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday

Week2: Monday 8th - Sunday 14th August

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday

Week3: Monday 15th - Sunday 21th August

Monday

Tuesday

Wednesday

Thursday
Nutritional Signal(Sugiura,Shimosaka & Okumura)
PCR Amplification of glnL and glnG from gDNA of E.coli

--Primers--
glnL
Left primer: tctagaggagactgctttatggcaac
Right primer: actagtaggaactatcgtcatcgactac
glnG
Left primer: tctagaggtgacgtttatgcaacga
Right primer: actagtacacacaagctgtgaatcactc
annealing temperature was 55 degrees.

lane 1,2 &7,8: DNA ladder(λDNA digested Hindlll,100bp), lane 3,4: glnG1,glnG2, lane 5,6: glnL1,glnL2

After purification, the concentration of DNA are
glnG1: 127.8ng/ul
glnG2: 118.1ng/ul
glnL1: 137.4ng/ul
glnL2: 124.2ng/ul

Friday
Nutritional Signal(Shimosaka)
Restriction of glnG1 and glnG2
Cut them with Xbal and Spel for 2 hours at 37 degrees.
Then, gel extraction of digested.

Saturday

Sunday

Week4: Monday 22th - Sunday 28th August

Monday

Tuesday

Wednesday

Thursday
Luminescence(Kusaba, Terada, Hara):ハエの走行性実験①　♂、紫外線×2回、緑×2回　♀、紫外線×2回、緑×2回

Friday
Luminescence(Kusaba):ハエの走行性実験①　♂、赤外線×2回　♀、赤外線×2回

Nutritional Signal(Hashiya): Transformation of bellow parts.
4-17M:BBa_K325909(lux operon)
1-12M:BBa_E0240
2-17F:BBa_120260(low copy vector)

PCR amplification of

Saturday
Luminescence(Kusaba):ハエの走行性実験①　♂、赤×2回、青×2回　♀、赤×2回、青×2回

Sunday

Week5: Monday 29th August - Sunday 4th September

Monday

Tuesday
Nutritional Signal(Hashiya)
・PCR amplification of glnL and glnG from PCR products,glnL1 and glnG1.
　--Primers--
　glnL
　Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac
　Right primer: ggactagtaggaactatcgtcatcgactac
　glnG
　Left primer: ggaattcgcggccgcttctagaggtgacgtttatgcaacga
　Right primer: ggactagtacacacaagctgtgaatcactc
　annealing temperature was 55 degrees.

・PCR amplification of glnL+G and rpoN from gDNA of E.coli.
　--Primers--
　glnL+G
　Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac
　Right primer: ggactagtacacacaagctgtgaatcactc
　rpoN
　Left primer: ggaattcgcggccgcttctagaggttctgaacatgaagcaa
　Right primer: ggactagtatccttatcggttgggtca
　annealing temperature was 56 degrees.

　
　lane1: 100bp DNA ladder, lane2:glnL, lane3:glnG, lane4:glnG+L, lane5:rpoN from gDNA, lane6:rpoN from ASKA clone
　　After purification, the concentration of DNA are
　glnL: 122.3 ng/ul
　glnG: 64.7 ng/ul
　glnL+G: 106.7 ng/ul
　rpoN from gDNA: 111.4 ng/ul

Wednesday
Nutritional Signal(Hashiya)
・Screening PCR of σ54 promoter + pSB1A3
　
　We cultured σ54 promoter5

Friday
Nutritional Signal(Hashiya)
・Restriction of σ54 promoter5,glnL, glnG, glnL+G and rpoN
　Cut them with EcoRl and Spel 　After purification, the concentration of DNA were
　σ54 promoter5: 23.6 ng/ul
　glnL: 28.1 ng/ul
　glnG: 26.3 ng/ul
　glnL+G: 15.3 ng/ul
　rpoN: 20.8 ng/ul

・Ligation reaction
　Ligated glnL, glnG and rpoN to pSB1K3.

Thursday
Nutritional Signal(Hashiya)
・Screening PCR of glnL, glnG, glnL+G and rpoN
glnL 　
glnG 　
glnL+G & rpoN 　
　We cultured glnL5, glnG4

Saturday
Nutritional Signal(Hashiya)
・Mini prep of glnL5 and glnG4
　glnL5: 43.9 ng/ul
　glnG4: 38.1 ng/ul

・Screening PCR of rpoN 　

Predation(Hashiya)
・PCR amplification of glmS
　--Primers--
　left primer:ggaattcgcggccgcttctagagcaggttgaccgacaacgata
　right primer:ggactagtacgcagggcatccatttat
　
　lane1: 100bp DNA ladder, lane2: glmS from gDNA, lane3: glmS from ASKA clone

・TA cloning of glmS
　Ligated glmS from gDNA to pTA vector.

Sunday
Nutritional Signal(Hashiya)
・Screening PCR of rpoN
　

・Transformation of bellow parts
　1-23L: BBa_B0015 (double terminator)
　1-18E: BBa_J23101 (constitutive promoter)
　1-18C: BBa_J23100 (constitutive promoter)

Week6: Monday 5th September - Sunday 11th September

Monday
Nutritional Signal(Hashiya)
・Screening PCR of glnL+G+double terminator

Tuesday
Luminescence(Kusaba, Hara):ハエの走行性実験②（②は改良版）　♂、緑×2回　♀、青×1回

Wednesday
Luminescence(Hara):ハエの走行性実験②　♂、青×2回　♀、緑×2回、青×1回

Thursday
Luminescence(Hara):ハエの走行性実験②　♂、紫外線×3回　♀、紫外線×3回

Friday

Saturday
Luminescence(Kusaba, Hara):ハエの走行性実験②　♂、青×2回、赤×2回　♀、青×2回、赤×2回　

Sunday
Luminescence(Kusaba, Hara):ハエの走行性実験②　♂、紫外線×2回、

Week7: Monday 12th September - Sunday 18th September

Monday

Luminescence:大腸菌の形質転換(Hashiya)　ハエの走行性実験②(Kusaba, Hara)

Tuesday

Luminescence:大腸菌はじめて光る。しかし光量は少ない。

Wednesday

Thursday

Friday

Digestion(Kajita)

PCR amplification of SAM-P20 and ChiA

We performed colony direct PCRs from a S. albogriseolus colony and a S. avermitilis colony.

Reaction mixture
Component	Volume(μl)
2x Buffer	25
2mM dNTPs	10
Primer 1	1.5
Primer 2	1.5
Template	X
KOD FX	1
ddH₂O	up to 50

PCR condition
Predenature	94C	2m
Denature	98C	10s	30cycles
Annealing	56C	30s
Extension	68C	1m30s

We prepared two kinds of templates:

Picked a colony, suspended in 50ul of water and incubated 1min at 95 degree. Added 1ul to the reaction mixture.
Picked a colony and dipped in the reaction mixture.

Gel electrophoresis indicated that the PCR amplifications were successful for a sample, ChiA, but it was a faint band. We decided to retry direct PCR of SAM-P20 and PCR of ChiA.

Saturday

Digestion(Kajita) Retry of PCR amplification of SAM-P20 and ChiA.

We amplified SAM-P20 by colony direct PCR and ChiA by PCR using the PCR product that performed yesterday.

Reaction mixture: The same components and volume as before.

PCR condition: The same PCR condition as before.

PCR for SAM-P20

We prepared two kinds of templates:

Picked a colony, suspended in 50ul of TE buffer (pH8.0) and incubated 1min at 95 degree. Added 5ul to the reaction mixture.
Picked a colony and dipped in the reaction mixture.

PCR for ChiA

1μl of PCR product was added to the reaction mixture as template.

Sunday

Week8: Monday 19th September - Sunday 25th September

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday

Week9: Monday 26th September - Sunday 2nd October

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday

Protocol

Medium for drosophila

[http://www.biol.se.tmu.ac.jp/fly/www/standard-medium.html Medium for drosophila]
Materials	Methods
water : 500mL dry yeast : 20g corn flour : 45g glucose : 50g agarose : 3.5~5g propionic acid : 1.5mL 10% p-hydroxybenzoate in 70% Eternol : 5g	Stir dry yeast and agarose with about two-thirds of water. Then, autoclave it. Stir corn flour and glucose with the remaining water. Stir 1 and 2, then autoclave it again. after autoclave, add propionic acid and 10% p-hydroxybenzoate in 70% Eternol into it.■

@@ Line 297: / Line 297: @@
 :{|class="wikitable"
 |+Reaction mixture
-! width="80"|Component
+!Component||Volume(&mu;l)
-!width="80"|Volume(&mu;l)
 |-
 |2x Buffer||25
@@ Line 309: / Line 308: @@
 |-
 |Template||X
+|-
+|KOD FX||1
 |-
 |ddH<sub>2</sub>O||up to 50
@@ Line 316: / Line 317: @@
 |Predenature||94C||2m||
 |-
-|Denature||98C||10s||rowspan="3"|25cycles
+|Denature||98C||10s||rowspan="3"|30cycles
 |-
 |Annealing||56C||30s
@@ Line 331: / Line 332: @@
 '''Saturday'''<br/>
+Digestion(Kajita)
+Retry of PCR amplification of SAM-P20 and ChiA.
+:We amplified SAM-P20 by colony direct PCR and ChiA by PCR using the PCR product that performed yesterday.
+:Reaction mixture: The same components and volume as before.
+:PCR condition: The same PCR condition as before.
+:PCR for SAM-P20
+:We prepared two kinds of templates:
+#Picked a colony, suspended in 50ul of TE buffer (pH8.0) and incubated 1min at 95 degree. Added 5ul to the reaction mixture.
+#Picked a colony and dipped in the reaction mixture.
+:PCR for ChiA
+:1&mu;l of PCR product was added to the reaction mixture as template.
 '''Sunday'''<br/>

Team:Kyoto/Lab Work

From 2011.igem.org

Revision as of 16:54, 22 September 2011

Contents

Lab Work

Week1: Monday 1st - Sunday 7th August

Week2: Monday 8th - Sunday 14th August

Week3: Monday 15th - Sunday 21th August

Week4: Monday 22th - Sunday 28th August

Week5: Monday 29th August - Sunday 4th September

Week6: Monday 5th September - Sunday 11th September

Week7: Monday 12th September - Sunday 18th September

Week8: Monday 19th September - Sunday 25th September

Week9: Monday 26th September - Sunday 2nd October

Protocol

Medium for drosophila