Plasmid Purification

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Reagents and Materials

1. Add 2 ml of culture medium of E.coli which cultured in overnights into an Eppendorf tube. Then, centrifuge the tube in room temperature (23~25℃), 130000 rpm for one minute.
2. Add 250 μl Buffer P1 into the tube. Then, stir it with Vortex until deposition disappears.
3. Add 250 μl Buffer P2 and mix the contents by inverting the tube (Don’t use the Vortex)
4. Add 350 μl Buffer N3 and mix the contents by inverting the tube (Don’t use the Vortex.
5. Centrifuge the tube in room temperature, 13000 rpm for 10 minutes.
6. Move the supernatant by a pipette from the tube to the QIAprep Spin Column.
7. Centrifuge in room temperature, 13000 rpm for 1 minute and throw away the flow through liquid which collected at the bottom of the column.
8. Add 500 μl Buffer PB and centrifuge the tube in room temperature, 13000 rpm for 1 minute. Then, throw away the flow through liquid.
9. Add 500 μl Buffer PE and centrifuge the tube in room temperature, 13000 rpm for 1 minute. Then, throw away the flow through liquid.
10. Set the upper part of the QIAprep Spin Column into another Eppendorf tube.
11. Add 50 μl EB buffer and left it at room temperature for 1 minute.
12. Centrifuge in room temperature, 13000 rpm for 1 minute. The flow through liquid is the plasmid DNA solution.

Restriction Enzyme Digestion

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Reagents and Materials

Eppendorf tube

milliQ

DNA solution

Digest Buffer

Restriction enzyme

Mix these solutions with the following quantities.

Pattern1 milliQ 12 µl DNA solution 5 µl Difest Buffer 2 µl Restriction enzyme 1 µl total 20 µl

Pattern 2 milliQ 12 µl DNA solution 5 µl Difest Buffer 2 µl Restriction enzyme ① 1 µl Restriction enzyme ② 1 µl total 20 µl

1. Incubate them at 37℃ for 1 hour.
2. Incubate them at 60℃ for 15 minutes.

Electrophoresis

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Reagents and Materials

Agarose gel

TAE Buffer

Loading Buffer

DNA solution

Eppendorf tube

Ethidium bromide

1. Set gel in the electrophoresis tank anf pour the TAE Buffer into the tank.
2. Put DNAsolution and Loading Buffer in the ratio of 1:2 into the Eppendorf tube and mix them.
3. Load samples into wells.
4. Do Electrophoresis at 100V for 40min.
5. After electrophoresis, soak the gel into water including ethidium bromide for 20min.
6. Observe the bands by ultraviolet radiation.

DNA Purification (PCR / Digest)

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Reagents and Materials

NusleoSpin Gel and PCR Clean-up

PCR / Digest solution

Centrifuge machine

Eppendorf tuber

1. Put NT1 Buffer and gel into a tube and warm in at 50℃ for 5 minutes and dissolve it. (Add 200 μl NT1 Buffer for 100 mg of gel)
2. Set the column into the Collection Tube and pour “Step1 solution” this column.
3. Centrifuge it at 4℃ at 11,000rpm for 30seconds and throw away the flow through solution and set the column again.
4. Add 700 µl Wash Buffer NT3 into the column and centrifuge it at 4℃ at 11,000 rpm for 30seconds.
5. Through away the flow through solution and set the column again.
6. Centrifuge at 4℃ at 11000 rpm for 1 minute.
7. Set the column in to the micro tube and add 43 µl NE Buffer into the column.
8. Left it for 1min at room temperature.
9. Centrifuge at 4℃ at 11000rpm for 1 minute.

PCR

1. Switch on the thermal cycler.
2. Make Cell suspension.
3. Put the following solution into the PCR tube (Total: 50 μl)

dNTP	4 µl
Primer(forward)	5 µl
Primer(reverse)	5 µl
Buffer	10 µl



4. Add 25 µl Cell suspension to the PCR tube.
5. Add 1µl Taq polymerase and stir thecontents with Vortex mixer.




P1 phage preparation

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1. ① making calcium cultures
• overnight culture 0.5ml
• LB midium （liquid） 1.4ml
• CaCl2 - 0.1M 0.1ml

Cultivation condition （37℃, 130 shake/minute, over 2 hour）



2. ② making LB・calcium plates
   • DW 100ml
   • Trypton 1g
   • Yeast Extract 0.5g
   • NaCl 1g
   • NaOH-10N 20ml
   • agarose 1g



Autoclave (121℃, 25 minutes ).

add CaCl2 0.1M 0.5ml

Dispense this midium in 4 plates.



3. ③ Preparation of　soft agar
   1. prepare a alminium thermostat block and soft agar（agarose 0.3％）
   2. Warm soft agar on alminium thermostat block
   3. Dispense 3ml soft agar in a test tube.
   4. We move test tubes in a alminium block.


4. ④ superposition of soft agar
• To transfuse Ca culture(0.4ml) into the test tube.
• To add IPTG(5µl) and P1 bacteriophage (2µl) to the test tube.
• To mix Soft agar well by a vortex mixer.
• Soft agar pour to the test tube which contains caculture(0.4ml) and IPTG(5µl).
• To mix test tube well by a mixer and poured into Ca・LB(solid)
• After the content set, it is incubated with an incubator for 6 ~ 7 hours.
• We prepare chloroform and we put chloroform 0.1 cc into a centrifugal tube.
• After it is centrifuged, its supernatant is moved to the centrifugal tube containing chloroform and they are mixed by mixer..
• The centrifuge is carried out.(speed: 3000・10min・ACCEL 8・４degrees )
• The supernatant is moved to the newly test tube.
• It is saved in a refrigerator of 4 degrees.



5. ⑤ soft agarのかきだし
1. Soft agar is gathered up in one side using a spreader.
2. Soft agar is moved to the test tube using a Pipetman.
3. The test tube is mixed by a mixer so that soft agar is powderized
4. Sentrifuge is carried out (speed 3000・10min・ACCEL8・4 degrees)

Electroporation

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1. Putting LB 200ml into sakaguchi flask.
2. Putting IPTG 25mg in DW2ml.*1
3. Putting 0.5cc overnight culture and IPTG 2ml into the sakaguchi flask.
4. It is done shaking culture (37℃, 130 times).
5. It is put into the centrifuge tube and centrifuge(speed:8000, time:5, temperature:4).
6. When centrifugation finish, we discard the supernatant quickly.
7. A 10% glycerol of 10ml is put into it. And it also removes to other sentrifuge.
8. It is centrifuged(speed:4000, time:10min, temperature:4).>
9. After centrifugation finish, glycerol is scavenged with aspirator.
10. A glycerol of 10ml is put into it on ice and mix mildly.
11. A glycerol of 10ml is put into it again
12. It is centrifuge again(speed:4000, time: 10 min, temperature:4).
13. The DNA is on ice. And we put DNA 5λinto sample tube.
14. We put 1ml SOC medium into sampling tube, and put 5λIPTG.
15. When centrifugation finish, the supernatant is scavenged with aspirator.
16. We mix 10ml glycerol and suspend.
17. We mix 10ml glycerol again.
18. It is put into a centrifuge
19. It is centrifuge again(speed:4000, time: 10 min, temperature:4).
20. 5λIPTG is put into SOC..
21. When centrifugation finish, the supernatant is scavenged with aspirator.
22. We mix 40λwith 5λDNA which is introduction DNA in sampling tube and blend it using mixer. It is on ice.
23. All sample is put into cuvette for electroporation using a Pipetman.
24. Flow current to it
25. When we hear electric sounds, we put SOC in it.
26. We incubate it for an hour, 30℃.
27. We put it of 120λ per a plate. Also we mix IPTG of 5λper a plate. And we spread it.

(ア) *1 IPTG is for using RED recombination. RED recombination system is induced by IPTG in our strain. If you do electroporation for plasimid DNA, IPTG addition is unneeded.

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