Team:HokkaidoU Japan/Notebook/protocol

Notebook

main1

Protocol

Common

PCR (2 STEP)

ReagentVolume
template DNA1 µL
forward Primer 10 µM1 µL
reverse Primer 10 µM1 µL
KOD Plus NEO1 µL
KOD Plus NEO 10x Buffer5 µL
2 mM dNTPS5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturationnumber of cycle
Cycle 268℃elongation timeAnnealing / Elongationnumber of cycle
Store4℃HoldStore

PCR (3 STEP)

ReagentVolume
template DNA1 µL
forward Primer 10 µM1 µL
reverse Primer 10 µM1 µL
KOD Plus NEO1 µL
KOD Plus NEO 10x Buffer5 µL
2 mM dNTPS5 µL
25 mM MgSO43 µL
DW33 µL
Total50 µL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturationnumber of cycle
Cycle 2Tm value30 secAnnealingnumber of cycle
Cycle 368℃elongation timeElongationnumber of cycle
Store4℃HoldStore

Digestion

ReagentVolume
template DNA16 µL
restriction enzyme1 µL
restriction enzyme1 µL
buffer2 µL
Total20 µL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
260℃15 minInactivation
Store4℃HoldStore

Sequencing

ReagentVolume
template DNA1 µL
primer 1 µM1.5 µL
Ready Reaction Premix1 µL
5x Sequencing Buffer1.5 µL
DW5 µL
Total10 µL

Sequencing

StepTemp.TimeProcessCycle
Start96℃10 secDenaturation
Cycle 150℃5 sec-number of cycle
Cycle 260℃240 sec-number of cycle
Store4℃HoldStore

Ligation

ReagentVolume
vecter DNAvolume
insert DNAvolume
Mighty Mixvolume
DWvolume
Totalvolume

Ligation

StepTemp.TimeProcess
116℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

PCR Purification

FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
Purification of PCR products

Dephosphorylation

ReagentVolume
Dephosphorylating DNAvolume
Antarctic Phosphatasevolume
Antarctic Phosphatase Buffervolume
Totalvolume

Dephosphorylation

StepTemp.TimeProcess
137℃15 minDephosphorylation
265℃5 minInactivation
Store4℃HoldStore

Electrophoresis

Gel ConcentrationVoltageTimeBuffer
1% / 2%50 V / 100 V30 - 60 min1/2x TBE

Gel Extract

FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel

Annealing of Oligonucleotides

ReagentVolume
forward primer 10 µMvolume
reverse primer 10 µMvolume
NaClvolume
DW
Totalvolume

Annealing of Oligonucleotides

StepTemp.TimeProcess
1Tm value + 5℃30 secAnnealing
2-0.01℃ / secStabilization
Store4℃HoldStore

E. coli

Liquid Culture

ReagentVolume
Single Colony-
mediavolume
antibioticvolume

Culture for several hrs.

Mini-prep

FastGeneTM Plasmid mini kit (Nippon Genetics Co., Ltd)
fast / standard / low copy protocol

Colony PCR (2 STEP)

ReagentVolume
Single Colony-
forward primer 10 µMvolume
reverse primer 10 µMvolume
Kapa-Taqvolume
DWvolume
Totalvolume

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturationnumber of cycle
Cycle 272℃elongation timeAnnealing / Elongationnumber of cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Colony PCR

ReagentVolume
Single Colony-
forward primer 10 µMvolume
reverse primer 10 µMvolume
Kapa-Taqvolume
DWvolume
Totalvolume

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturationnumber of cycle
Cycle 230 secAnnealingnumber of cycle
Cycle 372℃elongation timeElongationnumber of cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

Transformation (with pre-culture)

  1. Add plasmid to thawed competent cells on ice.
  2. Place it on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Add LB.
  5. Incubate the cells for 2 hrs at 37℃.
  6. Spread 300 µL of the culture onto plate with antibiotic.
  7. Incubate the plate at 37℃.

Transformation (w/o pre-culture)

  1. Add plasmid to thawed competent cells on ice.
  2. Incubate on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Spread 300 µL of the culture onto plate with LBA.
  5. Incubate the plate at 37℃.

Ethanol Precipitation

  1. Add 1/10 volume of NaOAc, 1.5 µL of glycogen and 5/2 volume of 100% ethanol.
  2. Leave it at -80℃ for 1 hr.
  3. Centrifuge at 15,000 rpm for 15 min at 4℃.
  4. Remove supernatant and add 220 µL of 70% ethanol.
  5. Centrifuge at 15,000 rpm for 10 min at 4℃.
  6. Remove supernatant and air-dry at room temperature with light shield.
  7. Suspend with 10 µL of DW.

Streaking (Single Colony Isolation)

  1. Pick the colony with an inoculating loop from the agar plate.
  2. Drag the loop across on a new agar plate.
  3. Re-sterilise the loop and drag it across again.

Competent Cells

  1. Thaw original competent cells on ice.
  2. Add 5 µL of original competent cells to 2 mL of LB.
  3. Incubate the cells for 16 hrs at 37℃.
  4. Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.
  5. Incubate the cells at 130 rpm at 20℃, until OD600 reach 0.5.
  6. Take 50 mL of incubated cells to two differnt culture tubes and centrifuge them at 3,000 rpm for 20 min at 4℃.
  7. Remove supernatant and add 75 mL of TB to each tube.
  8. Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4℃.
  9. Remove supernatant and add 32 mL of TB.
  10. Add 32 µL of DMSO 10 times.
  11. Take 50 µL and freeze with liquid nitrogen.

L. casei

Preparation of Bacteria

  1. Added 5 mL of L. casei to MRS medium.
  2. Cultured overnight.
  3. Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
  4. Stand cultured for 1.5 ~ 2 hrs until OD600 is 0.4 ~ 0.5.
  5. Incubated the cells on ice for 10 min.
  6. Centrifuged at 6,000 g for 15 min at 4℃.
  7. Removed supernatant and added 30 mL of cooled PEB.
  8. Centrifuged at 6,000 g for 15 min at 4℃.
  9. Removed supernatant and added 30 mL of cooled PEB.
  10. Centrifuged at 6,000 g for 15 min at 4℃.
  11. Removed supernatant and added 1 mL of cooled PEB.

Electroporation

  1. Prepared the plasmid to 300 ng/10 µL (TE pH 8).
  2. Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.
  3. Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes(0.2 cm gap #1652086).
  4. Added 800 µL of cooled MRS medium and stand cultured for 3 hrs in 30℃.
  5. Spread on MRS plate (Em 5 µg/mL).

Buffer

PEB Buffer

ReagentVolume
0.1 M Phosphate Buffer (pH 7.3)0.7 mL
Sucrose4.9 g
Total100 µL

Filtrate with 0.22 µm filter.

0.1 M Phosphate Buffer

Mix 0.1 M NaH2PO4 and 0.1 M Na2HPO4 and prepare it to pH 7.3..
Autoclave for 20 min at 121℃.

←Notebook Top

Link to iGEM HokkaidoU website main1 Link to iGEM HokkaidoU twitter
Link to iGEM HokkaidoU facebook page main5