Protocols

Transformation

1. Added (1~2) µL of (DNA) to (50) µL of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Added (600) µL of LB.
4. (Incubated the cells for 2 hrs at 37C.)
5. Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
6. (Added 900 µL of LB to 100 µL of the culture and spread 300 µL of it onto second plate.)
7. Incubated the plate(s) at 37C for 16~20 hours.

Mini-prep

Used mini-prep kit of Nippon genetics: FastGene Plasmid mini kit. This kit contains these reagents and wares: mP1 (resuspension buffer), mP2 (lysis buffer), mP3 (neutralization buffer), mP4 (first membrane wash buffer), mP5(second membrane wash buffer), mP6(elution buffer), column and collection tube.

1. Centrifuged (1~5) mL of culture at (over 10,000) rpm for 2 min.
2. Removed the supernatant.
3. Added 200 µL of mP1 and voltexed it.
4. Added 200 µL of mP2 and inverted the tube then left it for 2 min at room temperature.
5. Added 300 µL of mP3 then inverted the tube.
6. Centrifuged at 13,000 rpm for 2 min.
7. Loaded the supernatant to column tube.
8. Centrifuged at 13,000 rpm for 1 min.
9. Removed filtrate and added 400 µL of mP4 then centrifuged 13,000 rpm for 1 min.
10. Removed filtrate and added 600 µL of mP5 then centrifuged 13,000 rpm for 1 min.
11. Removed filtrate and centrifuged 13,000 rpm for 2 min.
12. Set column into 1.5 mL tube and added 50 µL of mP6.
13. Centrifuged at 13,000 rpm for 2 min.

Ethanol precipitation

1. Added (5) µL of NaOAc, 1.5 µL of glycogen and (125) µL of 100% ethanol.
2. (Left it at -80C for 1 hr. / Soaked liquid nitrogen in an instant.)
3. Centrifuged at 15,000 rpm for (10~15) min at 4C.
4. Removed supernatant and added (220) µL of 70% ethanol.
5. Centrifuged at 15,000 rpm for (5~15) min at 4C.
6. Removed supernatant and air-dried at room temperature with light sheilding.
7. Suspended with 10 µL of DW.

Ligation

Mixed the following reagents in 0.2 mL PCR tube. Used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer.

Solution	Vector DNA	Insert DNA	DW	Ligation 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

Digestion

Mixed the following reagents in PCR tube.

Solution	DNA	Restriction enzyme 1 10U/µL	Restriction enzyme 2 10U/µL	Appropriate buffer	DW	Total
Volume (µL)

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

Electrophoresis

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

Gel extraction

Used Gel extraction kit of Nippon genetics: FastGene Gel/PCR Extraction kit. This kit contains these reagents and wares: GP1 (binding buffer), GP2 (wash buffer), GP3 (elution buffer), column and collection tube.

1. Added 500 µL of GP1 to (~300 mg of) migrated gel and vortex.
2. Incubated the mixture at 55C for (10~15) min and inverted it.
3. Loaded the sample onto the column.
4. Centrifuged at 13,000 rpm for 1 min.
5. Removed filtrate and added 600 µL of GP2 and centrifuged at 13,000 rpm for 1 min.
6. Repeated step 5.
7. Removed filtrate and centrifuged at 13,000 rpm for 2 min.
8. Set column into 1.5 mL tube and added 50 µL of GP6.
9. Centrifuged at 13,000 rpm for 2 min.

PCR

Used KOD-Plus-Neo (TOYOBO) as polymerase. Mixed PCR solutions and ran the PCR machine in a program which is detailed below.

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

Thermal protocol is following

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

2STEP Cycle (Tm value ≥ 63C) Number Temp. Second 1 94 120 2 98 10 3 68 (30/kbp) 4 4 HOLD Cycle: 2~3 x (25~45) 3STEP Cycle (Tm value ≤ 63C) Number Temp. Second 1 94 120 2 98 10 3 (Tm value of primer) 30 4 68 (30/kbp) 5 4 HOLD Cycle: 2~4 x (25~45) cycleの右側の数字の意味がわかりにくいなと思いました Sequencing

Solution	template DNA	Forward Primer	Reverse		Total
Volume (µL)

Solution Volume( µL ) 5 x Sequencing Buffer　 1.5 ( primer) 1 uM 1.5 template DNA 1 Ready Reaction Premix 1 DW 5 Total 10 Number Temp. Second 1 96 10 2 50 5 3 60 240 5 4 HOLD Cycle: 2 x 25 Ethanol Presipitation Solution Volume( µL ) PCR product　　 10 DW 10 3 M NaOAc   2 Glycogen　　　 1 100% EtOH 50 centrifuged at 15,000 rpm for 15 min at room temprature Removed supernatant ,added 100 µL of 70% EtOH and tap tubes by finger. centrifuged at 15,000 rpm for 10 min at room temprature Removed supernatant and air dried at room temperature, after that 10 µL of  DW was added and dissolved the precipitate. Electrophoresis Resuspended the pellet to HiDi formamide and removed to 96-well plate. Set the plate and started electrophoresis. 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. Colony PCR Solution Volume( µL ) DNA solution(1 colony/10 µL DW) 4 Kapa-Taq(Taq polymerase) 10 10 uM EX-F primer 0.8 10 uM PS-R primer   0.8 DW 8.4 Total 20 Number Temp. Second 1 95 120 2 95 30 3 68.9 30 4 72 60 5 72 120 6 4 HOLD 2~4: 30~45 cycles

β－Galactosidase assay

(OZBIOSCHIENCES CPRG β－GAlactosidase Assay Kit : improved version)

1. Liquid culture transfected cells with a plasmid coding LacZ gene for 24 hrs in 2mL LB.
2. Centrifuge 100 µL of culture at 1000 G for 3 min.
3. Remove the supernatant and suspend the pellet in 50 µL of 5x Lysis Buffer.
4. Incubate the lysate for 15 min at room temperature by vortexing it several times to ensure complete lysis.
5. Add 50 µL of Standard Dilution Buffer to 13-well(changeable) of a 96-well plate, and make a standard curve.

β-Gal Standard (milliunits) Standard Dilution Buffer Volume β-Gal Standard Volume 20 999 µL 1 µL of β-gal standard stock 10 100 µL 100 µL of 20 mu β-gal standard 5 100 µL 100 µL of 10 mu β-gal standard 2.5 100 µL 100 µL of 5 mu β-gal standard 1.25 100 µL 100 µL of 2.5 mu β-gal standard 0.625 100 µL 100 µL of 1.25 mu β-gal standard 0.3125 100 µL 100 µL of 0.625 mu β-gal standard 0.15625 100 µL 100 µL of 0.3125 mu β-gal standard 0.078125 100 µL 100 µL of 0.15625 mu β-gal standard 0.0390625 100 µL 100 µL of 0.078125 mu β-gal standard 0.01953125 100 µL 100 µL of 0.0390625 mu β-gal standard 0.009765625 100 µL 100 µL of 0.01953125 mu β-gal standard 0 100 µL 1. Add 50 µL of each sample to new well. 2. Add 50 µL of 1x CPRG Substrate Solution to each well and incubate the plate at room temperature for about 2 hrs. 3. Add 100 µL of Stop Buffer to each well. 4. Monitor the color development(Measure the absorbance of each sample) at 570-595 nm. 5. Calculate the expression levels(the activity) of lacZ based on a standard curve