Team:Tokyo Metropolitan/Notebook

From 2011.igem.org

(Difference between revisions)
(Lab Protocols)
(PCR (PrimeSTAR® HS DNA Polymerase))
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==PCR (PrimeSTAR® HS DNA Polymerase)==
==PCR (PrimeSTAR® HS DNA Polymerase)==
*Ice
*Ice
-
*Micro pipette**Thermal cycler  
+
*Micro pipette
 +
*Thermal cycler  
*PCR Tube
*PCR Tube
*Micro Tube
*Micro Tube
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-
#Add the all following components on ice.
+
#Add following components on ice.
#*DW
#*DW
#*5× PCR Buffer
#*5× PCR Buffer
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#Dispense PCR solution to PCR tubes.
#Dispense PCR solution to PCR tubes.
#Set PCR tubes on thermal cycler
#Set PCR tubes on thermal cycler
-
#Run PCR  
+
#Run PCR
==Electrophoresis==
==Electrophoresis==

Revision as of 17:10, 4 October 2011

Tokyo Metropolitan Labnotes.png

Contents

Lab Notebook

Augast
Sun Mon Tue Wed Thu Fri Sat
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31
September
Sun Mon Tue Wed Thu Fri Sat
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30
Octorber
Sun Mon Tue Wed Thu Fri Sat
1
2 3 4 5 6

Lab Protocols

PCR (PrimeSTAR® HS DNA Polymerase)

  • Ice
  • Micro pipette
  • Thermal cycler
  • PCR Tube
  • Micro Tube
  • DW
  • 5× PCR Buffer
  • dNTP (2.5mM)
  • Primer(Forward & Reverse)  (20µM)
  • Template DNA
  • PrimeSTAR® HS DNA Polymerase (2.5U/μl)


  1. Add following components on ice.
    • DW
    • 5× PCR Buffer
    • dNTP (2.5mM)
    • Primer(Forward & Reverse)  (20µM)
    • Template DNA
    • PrimeSTAR® HS DNA Polymerase (2.5U/μl)
  2. Dispense PCR solution to PCR tubes.
  3. Set PCR tubes on thermal cycler
  4. Run PCR

Electrophoresis

  • Gel
  • Electrophoresis bath
  • Parafilm
  • Micro pipette
  • TAE Buffer
  • 6× Loading Buffer
  • DW
  • 5× PCR Buffer


  1. Set gel in a gel tank. Pour the TAE Buffer into the gel tank.
    • Loading Buffer
    • DNA solution
    • DW
  2. Mix these components on a Parafilm by pipetting.
  3. Pour the mixture sample into well.
  4. Switch on the power-source and run the gel at 100V for 20min.
  5. Observe the band by exposing ultraviolet radiation

Ethanol precipitation

  • Centrifuge
  • Micro Tube
  • Micro pipette
  • Aspirator
  • 99.5%Ethanol
  • 3.0M Sodium acetate (pH5.2)
  • TE Buffer


  1. Add 0.1 volume 3M Sodium acetate to the nucleic acid sample and vortex.
  2. Add 4 µl of Dr. GenTLETM Precipitation Carrier and vortex.
  3. Add 2.5 volumes of ethanol and vortex.
  4. Centrifuge at 12,000 rpm at 4˚C for 15 min.
  5. Discard the supernatant.
  6. Rinse the pellet with 70% ethanol and centrifuge again at 12,000 rpm at 4˚C for 5 min.
  7. Discard the supernatant and dry.
  8. Dissolve the pellet in sterilized water or TE buffer.

Plasmid Extraction

  • Ice
  • Micro Tube
  • Micro pipette
  • Centrifuge
  • 50mM glucose ; 10mM EDTA ; 25ml Tris-HCl (pH 8.0)
  • 5N NaOH
  • 10% SDS
  • 3M Potassium Acetate (pH 4.8)


  1. Centrifuge the E-coli pre-culture on 12,000 rpm for 5min, and then discard the supernatant.
  2. Add 100µl of the solution (50mM glucose; 10mM EDTA; 25ml Tris-HCl(pH 8.0)), and mix it gently
  3. Add 200µl of the 0.2N NaOH; 1% SDS, and mix it. Incubate on ice for 5min.
  4. Add 150µl of 3M Potassium Acetate (pH 4.8, 5M Acetic acid; 3M Potassium), and mix it. Incubate #on ice for 5min.
  5. Centrifuge on 12,000 rpm for 5min.
  6. Take supernatant.

Digestion

  • Micro Tube
  • Micro pipette
  • Heat Block or Water bus
  • Digest Enzyme
  • 10×Buffer 
  • DW


  1. Add below components into a tube and mix them.
    • DNA solution
    • DW
    • 10× Buffer
    • Digest enzyme
  2. Incubate at 37°C. from 2h to 16h

Ligation

  • Micro Tube
  • Micro pipette
  • Heat Block
  • T4 Ligase
  • 10× Buffer 
  • DW


  1. Add below components into a tube and mix them.
    • DNA solution 1
    • DNA solution 2   
    • DW
    • 10× Buffer
    • T4 Ligase
  2. Incubate at 16°C, from 30min to 1h

Transformation

  • Micro pipette
  • Heat Block or Water bus
  • ECOS™ Competent E. coli
  • Plate (Antibiotic)
  • Spreader


  1. Keep competent cells on ice to thaw it.
  2. Add plasmid DNA into E.coli cells.
  3. Incubate on ice for 5 min.
  4. Put tubes with DNA and E.coli into water bath at 42℃ for 45 seconds.
  5. Put tubes back on ice for 2 minutes.
  6. Add four times its volume of LB (with no antibiotic added). Incubate tubes for 30 min at 37℃.
  7. Spread about 100 ul of the resulting culture on LB plates
  8. Incubate overnight.

Colony PCR(Takara EX® Taq)

  • Plate
  • Ice
  • Micro pipette
  • Thermal cycler
  • Burner
  • 70%EtOH
  • Toothpick (爪楊枝)
  • PCRTube
  • Micro Tube
  • Spreader
  • DW
  • 10× PCR Buffer
  • dNTP (2.5mM)
  • primer(Forward & Reverse) (20µM)
  • Taq Polymerase
  1. Add the all following components on ice.
    • DW
    • 5× PCR Buffer
    • dNTP (2.5mM)
    • Primer(Forward & Reverse)  (20µM)
    • Template DNA
    • Taq Polymerase
  2. Dispense PCR solution to PCR tubes.
  3. Pick up a colony and put it into PCR tube.then inoculate to master plate.
  4. Set PCR tubes on thermal cycler
  5. Run PCR

Making Medium for Culture

  • Erlenmeyer flask
  • Graduated cylinder
  • Test Tube or Plate
  • Autoclave
  • Stirrer
  • Electronic balance
  • Burner or Clean bench
  • Yeast Extract
  • Peptone
  • NaCl
  • Antibiotic
  • Agar
  1. Measure component of medium and mix them in Graduated cylinder.
  2. Autoclave(121℃,20min).
  3. (Add appropriate antibiotics)
  4. Dispense medium to test tube or plate.
  5. Store at 4℃