Team:KIT-Kyoto/Notebook-Protocol

LB medium

*per kilogram

Bacto-tryptone	10g
Bacto-yeast extract	5g
NaCl	10g
dH2O	1000mL

1. Mix the reagents according to the previous components.
2. Adjust the pH (pH7.0) with NaOH.
3. Autoclave at 120℃ for 20min

LB plate

*per liter

Bacto-tryptone	10g
Bacto-yeast extract	5g
NaCl	10g
dH2O	1000mL

1. Mix the reagent according to the previous components.
2. Add NaOH and carry the enzymes pH7.0.
3. Add 15g Bacto agar and dissolve.
4. Autoclave at 120℃ 20min.
5. After cooling down approx. 60℃, add the suitable amounts of appropriate antibiotics.
6. Pour 20~30mL into laboratory dishes.

*Solution Ⅰ

Glucose	50mM
EDTA	10mM
Tris-HCl	25mM

*Solution Ⅱ

NaOH	0.2N
SDS	1%

*Solution Ⅲ

K-acetate

5M

Methods

Transformation into plasmid of E.coli.

1. Dissolve competent cell on ice.
2. Pour 100uL competent cell into 1.5mL tube, and mix with 1~5uL DNA.
3. Incubate for 30 minutes on ice.
4. Treat with heat shock the cells at 42℃ for 45 seconds.
5. Cool down on ice for 2 minutes.
6. Add 250uL S.O.C medium at room temperature.
7. Incubate at 37℃ with shaking for 1 hour.
8. Spread a suitable amount of the cells on LB plate (containing appropriate antibiotics).
9. Incubate overnight at 37℃.

Picking up a single colony

Pick up the colony grown on the LB plate by a platinum stick and put in 2.5mL of LB medium (containing appropriate antibiotics), and cultivate cells at 37˚C.

Miniprep by Alkaline-SDS method.

1. Spin 1.5 mL of culture in microcentrifuge tube at 12,000 rpm for 5 min. at 4 ˚C. Discard the supernatant
　and spin again for 2 min. Remove remaining liquid carefully.
2. Resuspend the bacterial cell pellet in 100 μL of solution I previously cooled on ice.
3. After 4 min. at room temperature, add 200 μL of solution II and mix by inverting several times.
4. Keep on ice for 4 min.
6. Add 160 μL of solution III and mix gently by inverting several times. Keep on ice for 5 min.
7. Centrifuge at 12,000 rpm for 10 min. at 4˚C
8. Transfer supernatant to new tube.
9. Add the same volume of phenol/chloroform and vortex well. Centrifuge at 12,000 rpm for 2 min at room
　temperature.
10. Transfer top clear layer to new tube. Add the same volume of diethyl ether and vortex. Centrifuge at
　12,000 rpm for 2 min. at room temperature.
11. Transfer the top clear layer to new tube. Add 2X volume of ethanol and vortex. Centrifuge at 12,000 rpm
　for 10 min. at room temperature.
12. Remove supernatant and add 1 mL of 70% ethanol and mix gently. Centrifuge at 12,000 rpm for 5 min at
　room temperature.
13. Remove supernatant and dry in vacuo. Dissolve pellet in 20 μL TE.

Miniprep by QIA prep Spin Miniprep Kit

1. Spin 1.5 mL of culture in microcentrifuge tube at 10,000 rpm for 5 min. at 4˚C. Discard the supernatant
　and spin again for 2 min. Remove remaining liquid carefully.
2. Resuspend the bacterial cell pellet in 250 μL of buffr P1.
3. Keep at room temperature for 4 min.
4. Add 250 μL of buffer P2 and mix by inverting quickly.
5. Keep on ice for 4 min.
6. Centrifuge at 13,000 rpm for 10 min. at 4˚C.
7. Apply supernatant to QIAprepspin column.
8. Centrifuge at 10,000 rpm for 40 sec. at room temperature. Discard the flow-through.
9. Add 500 μL of buffer PB. Centrifuge at 10,000 rpm for 40 sec. at room temperature. Discard the flow-
　through.
10. Add 750 μL of buffer PE. Centrifuge at 10,000 rpm for 40 sec. at room temperature. Discard the flow-
　through.
11. Centrifuge again at 10,000 rpm for 1 min. at room temperature. Set a column on new 1.5 mL tube.
12. Add 30 μL of buffer EB and keep at room temperature for 1 min. Centrifuge at 10,000 rpm for 1 min at
　room temperature.
13. Recover purified DNA in 1.5 mL tube.

Midiprep by Pure Link^TM HiPure Midiprep Kit

1. Centrifuge (4000xg,4°C,10min) E. coli culture in LB medium (50 mL) and remove the supernatant
2. Mix with suspension buffer (4mL) including RNase (20 micro g/mL) and the pellet
3. Add Lysis buffer (4mL) and turn upside and down several times in order to mix well
4. Incubate at room temperature for 5min
5. Add precipitation buffer (4mL) and shake the tube quickly
6. Centrifuge and remove the supernatant (15000 x g, room temperature, 10 min)
7. Transfer the supernatant to balanced column and elute out the solution by gravity
8. Wash the column twice with Wash buffer (10 mL) and elute out the solution as gravitation every after
　washing.
9. Put sterilized 15 mL centrifuge tube under the column
10. Add Elution buffer (5 mL) to the column and elute out the solution by gravity
11. Add isopropanol (3.5 mL) to the centrifuge tube and mix well
12. Centrifuge and remove the supernatant (15000 x g, 4°C, 30min)
13. Add 70% ethanol and mix well
14. Centrifuge and remove the supernatant (15000 x g, 4°C, 5min)
15. Dehydrated ,then suspend purified DNA into TE buffer (200 uL)

Purification of DNA from gel by QIA quick Gel Extraction Kit

1. Cut out the DNA fragment from agarose gel and put it in a 1.5mL tube and weigh the gel
2 .Add Buffer QG (x 3 volume of the gel weight)
3 .Vortex every 2 - 3 minutes to dissolve the gel completely
4 . Add isopropanol (equal weight to the gel, 100uL per 100mg)
5 . Trabsfer to spin column
6 . Centrifuge (10000rpm, room temperature,1min)
7 . Remove the solution
8 . Add Buffer PE (750 uL) and centrifuge (10000rpm,room temperature,1min)
9 . Remove the solution and centrifuge (17900 x g, room temperature, 1min)
10. Set a new 1.5 tube under the column, add TE (30uL) and centrifuge (10000 rpm, room temperature,1min)
11. Collect the purified DNA

BP reaction by Invitrogen gateway system

1. PCR using primers containing the attB sequence.
2. Purify PCR product.
3. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:

attB PCR product	75 ng/reaction (1-7 μL)
pDONR vector	150ng/reaction (1 μL)
TE Buffer	to 8 μL

4. Vortex BP ClonaseII enzyme mix briefly. Add 1 - 2 μL to the components above and mix well by vortexing
　and spin them down.
5. Incubate reaction at 25˚C for more than 1 hour.
6. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
7. Incubate at 37˚C for 10 minutes.

LR reaction by Invitrogen gateway system

1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:

Entry clones	50-150 ng/reaction (1-7 μL)
Destination vector	150ng/reaction (1 μL)
TE Buffer	to 8 − 9 μL

2. Vortex LR ClonaseII enzyme mix briefly. Add 1 – 2 μL, to the components above
　and mix well by vortexing and spin down.
3. Incubate reaction at 25˚C for 16 hours.
4. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly.
5. Incubate at 37˚C for 10 minutes.

Purification of plasmid DNA by High Pure PCR Product Kit

1. Add 500 uL of Binding buffer to DNA sample.
2. Apply the sample to the Spin column. Centrifuge the column at 20,000 x g for 1 min.
3. Discard the flow through fraction. Add 500 uL of Binding buffer to the column and centrifuge at 13,000 x g for 1 　　min.
4. Discard the flow through fraction. Add 200 uL of Binding buffer to the column and centrifuge at 13,000 x g for 1 　　min.
5. Discard the flow through fraction. Add 50 uL of Elution buffer to the column and centrifuge at 13,000 x g for 1 min.
　Eluted sample can be collected in 1.5 mL centrifuge tube. The purified plasmid DNA is recovered in this tube.

Protocol for Transfection of Adherent Cells (24 Well Plates)

1. The day before transfection, inoculate 24-well plates with an appropriate number of cells in serum-containing 　　　medium such that they will be 50 to 70% confluent the following day. For most cell lines, we recommend plating
　2.5×10⁵ cells in 0.5 ml of medium. Incubate the cells at 37℃ in 0％ incubator overnight.
2. Fifteen to sixty minutes prior to transfection, carefully aspirate the medium from the wells and add 250 µl of fresh 　growth medium to each well.
3. For each well to be transfected, prepare 25 µl of serum-free medium containing 1 pj of siLentFect as a starting 　　point.
4. For each well to be transfected, prepare 25 µl of serum-free medium containing DNA .Use a final concentration of 　10 nM as a starting point. For example, for a 24-well plate with 250 µl of growth medium per well, prepare 25 µl of 　serum-free medium containing 120 nM of DNA. After mixing with the diluted siLentFect from step 3 and addition to 　cells, the final concentration will be 10 nM. The optimal concentration of siRNA may vary from 5 to 20 nM 　　　　depending on the cell line used and the gene to be targeted.
5. Add the diluted DNA to the diluted siLentFect. Mix by tapping or pipetting.
　Incubate 20 minutes at room temperature.
6. Add 50 µl of complexes directly to cells in serum-containing medium. Rock the plate back and forth to mix.
　Incubate the cells at 37℃ in incubator.
7. Gene silencing can be monitored at the mRNA or protein levels from 4 to 72 hours after the transfection. If toxicity 　is a problem, change the medium 4 hours post transfection.

Embryo microinjection protochol

1. w; pΔ2,3 (female-virgin) yw (male)
　　- (prepare the flies from 3-4 days before)
2. mating
w; pΔ2,3 (female-virgin) X yw (male)
　　- (keep at 25℃ for 3-4 hrs ; usually from 9:00 AM)
3. prepare
　1) NaCl/Triton X-100, 10% Na-hypochloride, D.W. keep on ice.
　2) 50ml tube for embryo collection
　3) nylon mesh
　4) cover glass (3M tape, Cot No. W-18 pastes on the center)
　5) slide glass
　6) paraffin oil
　7) 1mg/ml DNA for micro injection
　8) glass needle
　9) Microinjection buffer
　　5 mM KCl, 0.1 mM Sodium Phosphate, pH6.8
　　DNA EtOH ppt, wash, dissolve to 1 mg/ml in the Microinjection buffe
4. collect the embryos
　　- wash 3-4 times with NaCl/Triton X-100
　　- dechorinate with 5 % Na-hypochloride
　　- wash 7-8 times with D.W.
5. put the dechorinated embryos on the prepared cover glass then drop parafin oil onto the embryos.
6. injection
7. move the tape containing the injected embryos to new egg plate.
　caution: the tapes keep upside down from avoiding dry for embryo.
8. after 1 day, move the hatched larva to new vials.
9. mating
　the hatched adult flies X yw female or male
10. take the flies having red eye then mate with yw.
　　- screening