Team:HokkaidoU Japan/Notebook/protocol

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Notebook

• Protocol

• E. coli
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Protocol

Common

PCR (2 STEP)

Reagent	Volume
template DNA	1 μL
forward Primer 10 μM	1 μL
reverse Primer 10 μM	1 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	number of cycle
Cycle 2	68℃	elongation time	Annealing / Elongation	number of cycle
Store	4℃	Hold	Store

PCR (3 STEP)

Reagent	Volume
template DNA	1 μL
forward Primer 10 μM	1 μL
reverse Primer 10 μM	1 μL
KOD Plus NEO	1 μL
KOD Plus NEO 10x Buffer	5 μL
2 mM dNTPS	5 μL
25 mM MgSO4	3 μL
DW	33 μL
Total	50 μL

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	94℃	120 sec	Initialization
Cycle 1	98℃	10 sec	Denaturation	number of cycle
Cycle 2	Tm value	30 sec	Annealing	number of cycle
Cycle 3	68℃	elongation time	Elongation	number of cycle
Store	4℃	Hold	Store

Digestion

Reagent	Volume
template DNA	16 μL
restriction enzyme	1 μL
restriction enzyme	1 μL
buffer	2 μL
Total	20 μL

Digestion

Step	Temp.	Time	Process
1	37℃	120 min	Digestion
2	60℃	15 min	Inactivation
Store	4℃	Hold	Store

Sequencing

Reagent	Volume
template DNA	1 μL
primer 1 µM	1.5 μL
Ready Reaction Premix	1 μL
5x Sequencing Buffer	1.5 μL
DW	5 μL
Total	10 μL

Sequencing

Step	Temp.	Time	Process	Cycle
Start	96℃	10 sec	Denaturation
Cycle 1	50℃	5 sec	-	number of cycle
Cycle 2	60℃	240 sec	-	number of cycle
Store	4℃	Hold	Store

Ligation

Reagent	Volume
vecter DNA	volume
insert DNA	volume
Mighty Mix	volume
DW	volume
Total	volume

Ligation

Step	Temp.	Time	Process
1	16℃	30 min	Ligation
2	65℃	10 min	Inactivation
Store	4℃	Hold	Store

PCR Purification

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

Dephosphorylation

Reagent	Volume
Dephosphorylating DNA	volume
Antarctic Phosphatase	volume
Antarctic Phosphatase Buffer	volume
Total	volume

Dephosphorylation

Step	Temp.	Time	Process
1	37℃	15 min	Dephosphorylation
2	65℃	5 min	Inactivation
Store	4℃	Hold	Store

Electrophoresis

Gel Concentration	Voltage	Time	Buffer
1% / 2%	50 V / 100 V	30 - 60 min	1/2x TBE

Gel Extract

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

Annealing of Oligonucleotides

Reagent	Volume
forward primer 10 μM	volume
reverse primer 10 μM	volume
NaCl	volume
DW
Total	volume

Annealing of Oligonucleotides

Step	Temp.	Time	Process
1	Tm value + 5℃	30 sec	Annealing
2	↓	-0.01℃ / sec	Stabilization
Store	4℃	Hold	Store

E. coli

Liquid Culture

Reagent	Volume
Single Colony	-
media	volume
antibiotic	volume

Culture for several hrs.

Mini-prep

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

Colony PCR (2 STEP)

Reagent	Volume
Single Colony	-
forward primer 10 μM	volume
reverse primer 10 μM	volume
Kapa-Taq	volume
DW	volume
Total	volume

2 Step Cycle (Tm value ≥ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	number of cycle
Cycle 2	72℃	elongation time	Annealing / Elongation	number of cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

Colony PCR

Reagent	Volume
Single Colony	-
forward primer 10 μM	volume
reverse primer 10 μM	volume
Kapa-Taq	volume
DW	volume
Total	volume

3 Step Cycle (Tm value ≤ 63℃)

Step	Temp.	Time	Process	Cycle
Start	95℃	120 sec	Initialization
Cycle 1	95℃	30 sec	Denaturation	number of cycle
Cycle 2		30 sec	Annealing	number of cycle
Cycle 3	72℃	elongation time	Elongation	number of cycle
Finish	72℃	120 sec	Final Elongation
Store	4℃	Hold	Store

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®/MicroPulser^TM 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

Reagent	Volume
0.1 M Phosphate Buffer (pH 7.3)	0.7 mL
Sucrose	4.9 g
Total	100 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℃.

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