Team:UT-Tokyo/LabWork/RegularMethods

From 2012.igem.org

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<!-- 以下テンプレ部分まで自由記述 -->
<!-- 以下テンプレ部分まで自由記述 -->
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==H<sub>2</sub> detection and assay ==
+
==Assembly parts ==
 +
===Digestion===
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===Gas Chromatography===
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====Materials====
 +
*Plasmid
 +
*10x buffer
 +
*100x Acet BSA
 +
*Emzyme (EcoR I, Xba I, Spe I, Pst I)
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Gas Chromatography is a method used for separating and analyzing compounds by injecting ａ sample mixture into a column prepared to be in a suitable stationary phase and passing a gas （carrier gas） in the mobile phase through the column, in order to separate the mixture into its components by making use of the difference in retention capacity against the stationary phase. This method can be applied to a gaseous or vaporizable sample, and is used for identification, purity testing, and quantitative determination.
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<br>
 +
====Protocol====
 +
1. Add plasmid
 +
*MilliQ up to 20uL
 +
*2uL 10x H or M buffer
 +
*0.2uL BSA
 +
*0.5uL emzyme I
 +
*0.5uL emzyme II
 +
<br>
 +
2. Incubation at 37 °C for more than two hour.
 +
<br>
-
===Materials===
+
===Ligation===
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*Gas Chromatograph ; SHIMADZU GC-8A with a thermal conductivity detector
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====Materials====
 +
*Vector DNA
 +
*Insert DNA
 +
*2x Ligation Mix
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Column ; molecular sieve 13X 60/80
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====Protocol====
 +
1. Make reaction liquid
 +
*MilliQ up to 20uL
 +
*10uL 2x Ligation Mix
 +
*Vector DNA
 +
*Insert DNA
 +
<br>
 +
2. Incubation at 16 °C for 15-30 min.
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Carrer gas ; nitrogen at 30 mL/min
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==Transformation==
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Injection temperature ; 50℃
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====Materials====
 +
*BioBrick parts / ligation products
 +
*SOC or LB (No antibiotic) 500uL
 +
*TE 15uL
 +
*plates
 +
*competent cells
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Column temperature ; 42℃
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====Protocol====
 +
to thaw out igem parts
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Current ； 90 mA
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1. With a pipette tip, punch a hole in the foil
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*0.5 mL micro-syringe
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2. Add 15uL of TE (MilliQ),and pipetting
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*2 mL vial
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*Single colony of bacteria containing our construct that raises hydrogen production;
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FhlA      ; Plac－RBS－FhlA-d.term
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m-FhlA ; Plac－RBS－(m-FhlA)－d.term
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3. Pipette 1uL of the resuspended DNA Transformation into your desired competent cells
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==Protocol==
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4. Hold on ice for 30 min.
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1. A single colony of cells transformed with engineered plasmids (FhlA, m-FhlA) was inoculated into 2 mL of LB with appropriate antibiotics and grown to saturation at 37°C.
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2. 100 μL of The saturated culture was added to  fresh LB broth and grown till the OD<sub>600</sub> = 0.4.
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5. Heat shock at 42°C for 45 seconds (and on ice after it)
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3. The culture was induced with 1 mM IPTG at 37°C for 1 hour. Then, formic acid was added so that its final concentration was 0 mM, 20 mM, or 60 mM. 
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6. Add 300uL of LBborth in each epp
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4. 1mL LB broth  was accurately added to a 2 mL vial. Then, gaseous phase  substitution was carried out using nitrogen.
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7. Wait for 10 mins
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5. It was left to stand at 37℃ for 8 hours
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8. Hold at 37°C for 30 min.
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6. 0.3 mL of the sample　from the gaseous phase was injected into the gas chromatograph.
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　(this step can be skipped with ampicillin selection)
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7. The peak area of the hydrogen obtained from gas chromatography was read, and the amount of hydrogen generated was calculated.  
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9. Plate out
 +
 
 +
10. Incubate at 37°C
 +
 
 +
==Purification of DNA==
 +
 
 +
===Miniprep===
 +
 
 +
====Material====
 +
*kit of Promega (SVMinipreps)
 +
*incubative tube
 +
*1.5mL epp tube
 +
*MilliQ
 +
====Protocol====
 +
1. pour contents out of the incubative tube into the 1.5mL tube as you can
 +
 
 +
2. centrifuge for 10min (15,000rpm)
 +
 
 +
　(you can centrifuge incubative tube directly when it can endure up to 6,000g )
 +
 
 +
3. throw supernatant fluid away not to damage the precipitation
 +
 
 +
　( you should decant by using yellow tip first / remove culture medium as you can / throw waste water away in bio hazard!)
 +
 
 +
4.add 250uL cell resuspension solution (red label)、suspend completely
 +
 
 +
　(incomplete suspending decreases yields / you should use epp stand like a washboard)
 +
 
 +
5. add 250uL Cell lysis solution（green label）
 +
 
 +
6. turn the tube upside down four times slowly not to bubble
 +
 
 +
7. add 10uL Alkalin　Protease　Sol. （small bottle）
 +
 
 +
8. turn the tube upside down four times slowly not to bubble
 +
 
 +
9. wait for 5min （Be careful not to exceed 5min! colon bacillus will disintegrate too much!）
 +
 
 +
10. add 350uL Neutralization Sol. (blue label）
 +
 
 +
11. turn the tube upside down four times slowly not to bubble
 +
 
 +
12. centrifuge for 10min (15,000rpm)
 +
 
 +
13. put the supernatant fluid to column (germ’s wreckage is adhering below)
 +
 
 +
14. centrifuge for 1min (15,000rpm)
 +
 
 +
15. throw flow through (the liquid in the tube below) away
 +
 
 +
16. add 750uL Wash Sol. to column and centrifuge for 1min (15,000rpm)
 +
 
 +
17. throw flow through away, put 250μL Wash Sol. to column and centrifuge for 1min (15,000rpm)
 +
 
 +
18. change the column into 1.5ml tube and centrifuge for 2min (15,000rpm)
 +
 
 +
19. change the tube into new one and add 50uL MilliQ
 +
 
 +
　(use Nucleas-Free Water in the kit instead of MilliQ)
 +
 
 +
20. centrifuge for 1min (15,000rpm) after waiting for 1min
 +
 
 +
21. take 1 to 1.5uL and determine the concentration by NanoDrop (Don’t dilute)
 +
 
 +
22. label them
 +
 
 +
<br>
 +
 
 +
===Gel extraction, PCR clean-up===
 +
 
 +
====Material====
 +
*kit of Promega (SVMinipreps)
 +
*Gel
 +
 
 +
====Protocol====
 +
1. Gel Slice and PCR Product Preparation
 +
 
 +
　Dissolving the Gel Slice
 +
　*Cut out gel with wanted band and put it in a tube.
 +
　*Add 3 parts Mem. binding sol. to 1 part Gel volume.
 +
 
 +
　Processing PCR Amplifications
 +
　*Add an equal volume of Membrane Binding Solution to the PCR amplification.
 +
 
 +
2. Shake & Incubate at 50-65 degrees C until gel is completly dissolved.
 +
 
 +
3. Put in the column.
 +
 
 +
4. Centrifuge at 15,000 rpm for 1 minute
 +
 
 +
5. Empty collection tube and add 700 ul Mem. Wash sol, centrifuge for 1 minute.
 +
 
 +
6. Empty collection tube and add 500 ul Mem. Wash sol, centrifuge for 1 minute.
 +
 
 +
7. Change the column to a new 1.5 ml Eppendorf and centrifuge at 15,000 rpm for 1 minute.
 +
 
 +
8. Change the old tube to a new 1.5 ml Eppendorf, add 30ul Nuclease-Free Water, incubate at RT for 1 minutes, then centrifuge at 15,000 rpm for 1 minute.
 +
 
 +
9. Measure concentration, label the Eppendorf.
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{{:Team:UT-Tokyo/Template/Footer|prevname=Lab Work|prevfull=LabWork|nextname=Assay Methods|nextfull=LabWork/AssayMethods}}
{{:Team:UT-Tokyo/Template/Footer|prevname=Lab Work|prevfull=LabWork|nextname=Assay Methods|nextfull=LabWork/AssayMethods}}

Latest revision as of 00:09, 27 September 2012

Regular Methods

Assembly parts

Digestion

Materials

  • Plasmid
  • 10x buffer
  • 100x Acet BSA
  • Emzyme (EcoR I, Xba I, Spe I, Pst I)


Protocol

1. Add plasmid

  • MilliQ up to 20uL
  • 2uL 10x H or M buffer
  • 0.2uL BSA
  • 0.5uL emzyme I
  • 0.5uL emzyme II


2. Incubation at 37 °C for more than two hour.


Ligation

Materials

  • Vector DNA
  • Insert DNA
  • 2x Ligation Mix

Protocol

1. Make reaction liquid

  • MilliQ up to 20uL
  • 10uL 2x Ligation Mix
  • Vector DNA
  • Insert DNA


2. Incubation at 16 °C for 15-30 min.

Transformation

Materials

  • BioBrick parts / ligation products
  • SOC or LB (No antibiotic) 500uL
  • TE 15uL
  • plates
  • competent cells

Protocol

to thaw out igem parts

1. With a pipette tip, punch a hole in the foil

2. Add 15uL of TE (MilliQ),and pipetting

3. Pipette 1uL of the resuspended DNA Transformation into your desired competent cells

4. Hold on ice for 30 min.

5. Heat shock at 42°C for 45 seconds (and on ice after it)

6. Add 300uL of LBborth in each epp

7. Wait for 10 mins

8. Hold at 37°C for 30 min.

　(this step can be skipped with ampicillin selection)

9. Plate out

10. Incubate at 37°C

Purification of DNA

Miniprep

Material

  • kit of Promega (SVMinipreps)
  • incubative tube
  • 1.5mL epp tube
  • MilliQ

Protocol

1. pour contents out of the incubative tube into the 1.5mL tube as you can

2. centrifuge for 10min (15,000rpm)

　(you can centrifuge incubative tube directly when it can endure up to 6,000g )

3. throw supernatant fluid away not to damage the precipitation

　( you should decant by using yellow tip first / remove culture medium as you can / throw waste water away in bio hazard!)

4.add 250uL cell resuspension solution (red label)、suspend completely

　(incomplete suspending decreases yields / you should use epp stand like a washboard)

5. add 250uL Cell lysis solution（green label）

6. turn the tube upside down four times slowly not to bubble

7. add 10uL Alkalin　Protease　Sol. （small bottle）

8. turn the tube upside down four times slowly not to bubble

9. wait for 5min （Be careful not to exceed 5min! colon bacillus will disintegrate too much!）

10. add 350uL Neutralization Sol. (blue label）

11. turn the tube upside down four times slowly not to bubble

12. centrifuge for 10min (15,000rpm)

13. put the supernatant fluid to column (germ’s wreckage is adhering below)

14. centrifuge for 1min (15,000rpm)

15. throw flow through (the liquid in the tube below) away

16. add 750uL Wash Sol. to column and centrifuge for 1min (15,000rpm)

17. throw flow through away, put 250μL Wash Sol. to column and centrifuge for 1min (15,000rpm)

18. change the column into 1.5ml tube and centrifuge for 2min (15,000rpm)

19. change the tube into new one and add 50uL MilliQ

　(use Nucleas-Free Water in the kit instead of MilliQ)

20. centrifuge for 1min (15,000rpm) after waiting for 1min

21. take 1 to 1.5uL and determine the concentration by NanoDrop (Don’t dilute)

22. label them


Gel extraction, PCR clean-up

Material

  • kit of Promega (SVMinipreps)
  • Gel

Protocol

1. Gel Slice and PCR Product Preparation

　Dissolving the Gel Slice 　*Cut out gel with wanted band and put it in a tube. 　*Add 3 parts Mem. binding sol. to 1 part Gel volume.

　Processing PCR Amplifications 　*Add an equal volume of Membrane Binding Solution to the PCR amplification.

2. Shake & Incubate at 50-65 degrees C until gel is completly dissolved.

3. Put in the column.

4. Centrifuge at 15,000 rpm for 1 minute

5. Empty collection tube and add 700 ul Mem. Wash sol, centrifuge for 1 minute.

6. Empty collection tube and add 500 ul Mem. Wash sol, centrifuge for 1 minute.

7. Change the column to a new 1.5 ml Eppendorf and centrifuge at 15,000 rpm for 1 minute.

8. Change the old tube to a new 1.5 ml Eppendorf, add 30ul Nuclease-Free Water, incubate at RT for 1 minutes, then centrifuge at 15,000 rpm for 1 minute.

9. Measure concentration, label the Eppendorf.


Retrieved from "http://2012.igem.org/Team:UT-Tokyo/LabWork/RegularMethods"