Team:HokkaidoU Japan/Overview/Extra materials/Notebook/Prptocols

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Team:HokkaidoU Japan

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PCR

Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below.

Solution	template DNA	Primer-F 10µM	Primer-R 10µM	MgSO4	dNTPs	10x Buffer	KOD Plus Neo	DW	Total
Volume (µL)	1	1	1	3	5	5	1	33	50

Thermal protocol is following

2STEP Cycle (Tm value > 63°C)

Sequence	Temp. (°C)	Time (sec)
1	94	120
2	98	10
3	68	30sec / 1kbp
4	4	Hold

Cycle: sequence2~3 × (25~45)

3STEP Cycle (Tm value < 63°C)

Sequence	Temp. (°C)	Time (sec)
1	94	120
2	98	10
3	Tm	30
4	68	30sec / 1kbp
5	4	Hold

Cycle: sequence2~4 × (25~45)

STEP DOWN

Sequence	Temp. (°C)	Time (sec)
1	94	120
2	98	10
3	74	30sec / 1kbp
4	98	10
5	72	30
6	98	10
7	70	30
8	98	10
9	68	30
10	68	420
11	4	Hold

Cycle:sequence2~3 × (5)

sequence4~5 × (5)

sequence6~7 × (5)

sequence8~9 × (15)

PCR Purification

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

Digestion

Mix the following reagents in PCR tube.

Solution	DNA	RE1 10U/µL	RE2 10U/µL	Appropriate buffer	Total
Volume (µL)	16	1	1	2	20

Sequence	Temp. (°C)	Time (min)
1	37	120
2	65	15
3	4	Hold

Ligation

Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit <Mighty Mix&rt; (Takara Bio Inc.) which contains ligase and buffer.

Solution	Vector DNA	Insert DNA	DW	Mighty Mix	Total
Volume (µL)	1	2	2	5	10

Thermal protocol is following

Sequence	Temp. (°C)	Time (min)
1	16	30
2	65	10
3	4	Hold

Electrophoresis

1. Put gel into electrophoresis tank.
2. Pore 2x TBE buffer into the tank to soak gel.
3. Add 5 µL of EtBr into cathod.
4. Pre-migration for 30 min at 100 V.
5. Apply DNA solution with 6x loading dye and ladder.
6. Start electrophoresis at 100 V.

Gel Extraction

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

Ethanol precipitation

1. Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.
2. Leave it at room temperature for 5 min.
3. Centrifuge at 15,000 rpm for 15 min at 25°C.
4. Remove supernatant and add 600 µL of 70% ethanol.
5. Centrifuge at 15,000 rpm for 5 min at 25°C.
6. Remove supernatant and air-dry at room temperature with light sheilding.
7. Suspend with 10 µL of TE.

Colony PCR

Solution	DNA	Kapa-Taq (Taq polymerase)	EX-F primer 10µM	PS-R primer 10µM	Total
Volume (µL)	4.2	5	0.4	0.4	10

Sequence	Temp. (°C)	Time (sec)
1	94	120
2	94	30
3	68	60 sec / 1kbp
4	4	Hold

Cycles: sequence2~3 × 25~45

Sequencing

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

Sequence	Temp. (°C)	Time (sec)
1	96	10
2	50	5
3	60	240
4	4	Hold

Cycle: sequence2~4 × 25

Ethanol precipitation

Solution	PCR product	DW	3M NaOAc	Glycogen	100% EtOH
Volume (µL)	10	10	2	1	50

1. Centrifuge at 15,000 rpm for 15 min at room temprature
2. Remove supernatant ,add 100 µL of 70% EtOH and tap tubes by finger.
3. Centrifuge at 15,000 rpm for 10 min at room temprature
4. Remove supernatant and air dry at room temperature.
5. Resuspend the pellet to HiDi formamide and remove to 96-well plate.
6. Set the plate and start electrophoresis.

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°C.
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°C, 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°C.
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°C.
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.

Transformation

1. Add plasmid to thawed competent cells on ice.
2. Incubate on ice for 30 min.
3. Add to LB.
4. (Incubate the cells for 2 hrs at 37°C.)
5. Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
6. Incubate the plate(s) at 37°C for 16~20 hrs.

Mini-prep

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

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.
4. Repeat method 3.

PEG precipitation

1. Add 13 µL of PEG to 20 µL of product(s).
2. Leave it at room temperature for 1 hr.
3. Centrifuge at 15,000 rpm for 20 min at 4°C.
4. Remove supernatant and add 100 µL of 70% ethanol.
5. Centrifuge at 15,000 rpm for 2 min at 4°C.
6. Remove supernatant and air-dry at room temperature with light sheilding.
7. Suspend with 10 µL of TE.

Gel Free System

Preparation of biotinylated DNA fragments

Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase and 5'-biotinylated primers. Mix PCR solutions and run the PCR machine in a program which is detailed below.

Solution	template DNA	5'-biotinylated 100-UP primer 10µM	5'-biotinylated 200-DN primer 10µM	MgSO4	dNTPs	10x Buffer	KOD Plus Neo	DW	Total
Volume (µL)	1	1.5	1.5	3	5	5	1	32	50

Thermal protocol is following

2STEP Cycle (Tm value > 63°C)

Sequence	Temp. (°C)	Time (sec)
1	94	120
2	98	10
3	68	30sec / 1kbp
4	4	Hold

Cycle: sequence2~3 × (25~45)

Preparation of magnetic beads

1. Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.
2. Collect the beads by attracting them to one side in 0.2 mL polypropylene tube using neodymium magnet.
3. Remove supernatant.

Fixation to magnetic beads

1. Add 3 µL of PCR product (0.48 pmol) and 7 µL of TE to beads.
2. Mix by vibration using sonic-toothbrush.
3. Collect the beads using magnet.
4. Remove supernatant containing excess amount of free DNA fragment.

Double restriction digestion

1. Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease, XbaI and SpeI, to the beads.
2. Mix by pumping using pipette.
3. Incubate at 37 °C for 30 min.
4. Collect the beads using magnet.
5. Obtain supernatant containing digested DNA fragment.
6. Purify the supernatant by ethanol precipitation.