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Contents 1 Lab Notebook 2 Lab Protocols 2.1 PCR (PrimeSTAR® HS DNA Polymerase) 2.2 Electrophoresis 2.3 Ethanol precipitation 2.4 Plasmid Extraction 2.5 Digestion 2.6 Ligation 2.7 Transformation 2.8 Colony PCR(Takara EX® Taq) 2.9 Making Medium for Culture

Lab Notebook

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

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 into Microtube.

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 into water bath at 42℃ for 45 seconds.
5. Put tubes back on ice for 2 minutes.
6. Add four times 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
• 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
   • 10× 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 Erlenmeyer flask.
2. Autoclave(121℃,20min).
3. (Add appropriate antibiotics)
4. Dispense medium to test tube or plate.
5. Store at 4℃