Team:Kyoto/Protocols

Miniprep

• Use QIAprep Spin Miniprep Kit Cat. No. 27104 by QIAGEN

1. Pick a single colony from a freshly streaked selective plate and inoculate a culture of about 3ml LB medium containing the appropriate selective antibiotic.
2. Incubate at 170rpm for 8h at 37℃ with vigorous shaking.
3. Transfer a half of the culture to a tube.
4. Harvest the bacterial cells by centrifugation at 14,000 x g for 1min at 4℃. Remove the medium by decanting.
5. Transfer the half of the culture to same tube and harvest as same. Remove the medium by pippetting.
6. Resuspend pelleted bacterial cells in 250µl Buffer P1 and mix thoroughly by pippeting.
7. Add 250µl Buffer P2 and mix thoroughly by inverting the tube gently 4-6 times.
8. Add 350µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
9. Centrifuge for 10min at 14,000 x g at 4℃.
10. Apply the supernatants from step 10 to the QIAprep spin column by pipetting.
11. Centrifuge for 10s in a table-top microcentrifuge. Discard the flow-through.
12. Wash the QIAprep spin column by adding 0.5ml Buffer PB and centrifuging for 10s in a table-top microcentrifuge. Discard the flow-through.
13. Wash QIAprep spin column by adding 0.65ml Buffer PeE and centrifuging fo 10s in a table-top microcentrifuge.
14. Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.
15. Place the QIAprep column in a clean tube. To elute DNA, add 50µl water to the center of each QIAprep spin column, let stand for 1min, and centrifuge for 1min.
16. Discard the QIAprep spin column.
17. Measure the concentration of DNA by using eppendorf BioPhotometer plus.
18. Restriction Digestion.
19. Agarose Gel Electrophoresis for Confirmation.

PCR

1. Dilute template DNA. If the concentration of DNA is 2-100 ng/µL, transfer 1µl to a clean tube and add 99µl Milli-Q.
2. Dilute Primer. If the concentration of Primer is XµM, dilute primer X timers and transfer 1µl to a clean tube and add 99µl Milli-Q.
3. Make a solution as the below.
4. Let stand for 2min at 94℃.
5. 25-40 cycles for 10s at 98℃, for 30s at Tm-5℃, and for 1min (1min for 1kb) at 68℃ (Tm is temparature at which primer will dissolve).
6. At 15℃ forever.
7. Agarose Gel Electrophoresis for confirmation.

• * If amplification is not succeeded, try at 4.5 or 6.0µl 25mM MgSO4.

Gel Electrophoresis

Gel Extraction

• Use QIAquick Gel Extraction Kit

1. Transfer cutted gel to a tube.
2. Add BufferQC about 3 times as much as the volume of the gel.
3. Stand the gel for 10 min at 50℃ to Dissolve. If hard to dissolve, sometimes vortex.
4. Confirm the color of the solution. If the color is orange or purple, add about 10µl 3M sodium acetate to yellow.
5. Add isopropanol as much as the gel and mix.
6. Apply the solution to the column.
7. Centrifuge for 1 min at 1300 rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
8. Add 500 µl BufferQC and centrifuge for 1 min. Discard the through.
9. Add 750 µl BufferPE and let stand for 2-3 min. If the solution overflows, we decrease the amount of BufferPE.
10. Centrifuge for 1 min and Discard the through.
11. Centrifuge for additional 1 min to remove residual buffer.
12. Place the column in a clean tube.
13. Add 10 µl BufferEB or H20 to the center of each column, let stand for 1 min, and repeat this step.
14. Centrifuge for 1 min at 13000 rpm.
15. Discard the column.

Transformation

1. Unfreeze conpitent cells on ice.
2. Dry a plate by letting the plate upside down and partly open in incubator.
3. Add 1 µl DNA solution and 20µl the compitent cells to 1.5ml tube, let stand for 30min on ice.
   • If few colony is observed, increase the amount of the compitent cells or DNA, but make the amount of DNA not to get over that of the compitent cells.
4. Heatshock for 60s at 42℃.
5. Let stand for 2min on ice.
6. Cµlture for 1h in precµlture medium (LB or SOC medium), and plate by using spreader.
   • (Do not heat spreader too much because e.coli will dead for heat.

Making Media

1. Wash a graduated cylinder with MilliQ.
2. Add the following to about 180ml of MilliQ in the graduated cylinder:
   • Bacto-yeast extract : 1g final to 0.5% (w/v)
   • Trypthon : 2g final to 1% (w/v)
   • NaCl : 2g final to 1% (w/v)
   • 200µl 1N NaOH
3. Seal the graduated cylinder by a Parafilm.
4. Dissolve the mixture by inverting the graduated cylinder.
5. Adjust volume to 200ml by adding more MilliQ.
6. Add the media solution to a 200ml Erlenmeyer flask.
7. (To prepare solid media, add 2.4g (1.2%) of agar to the flask.)
8. Wrap the tops of the flasks with aluminum foil.
9. Place a small piece of auto clave tape on one of the flasks.
10. Autoclave the media on a liquids.
11. Store at room temperature.
12. To pour plates, melt the solid media in the microwave, then place flask in water bath to bring media to 50℃.
13. Add 200µl of antibiotic:
    • 100µg/ml for ampicillin
    • 50µg/ml for kanamycin
14. Add 0.5 - 1.0% Glucose for protein expression.

References

Bertani, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62:293-300. PMID 14888646

Making Agarose Gel

1. Add the following to a 200ml beaker.
   • Agarose : 2g
   • 1xTAE : 200ml
2. Wrap the tops of the beaker with plastic wrap.
3. Punch a hole through the wrap with a pipette tip.
4. Dissolve the gel by heating in a microwave and swirling without boiling.
5. Pour the gel into the Gel Maker.
6. If there is air bubbles, pushing them with a pipette tip.
7. Cover the Gel Maker with a plastic wrap.
8. Let stand for 45min.
9. Store in the Tupperware in the refrigerator.

Ligation

1. Create a Mixture (the vector and the insert at 1 : 5-10 molecµle) and a Control (only the vector).
2. Add Ligation High to create a solution.
3. Incubate at 16℃ for 30 min.

• If the colonies of E.coli transformed with the Control,

Screening PCR

1. Make a Mixture (Do on PCR Bench).
   • 10x PCR buffer (Boehringer) : 40µl
   • 2.5mM dNTP : 8µl
   • primer-1 (4pmole/µl) : 20µl
   • primer-2 (4pmole/µl) : 20µl
   • Taq polymerase (Boehringer) : 1.6µl
   • MilliQ : 310µl (to total 400µl)
2. Dispense 25µl x 15 tubes.
3. Pick a single colony and transfer it to each tubes.
4. Suspend the colony.
5. Let stand for 10min at 90℃.
6. 30 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.
7. Let stand for 4min at 72℃.
8. Add 5ml Loading Buffer to the tubes.
9. Agalose Gel Electrophoresis for confirmation.
   • Negative Control : Use nothing.
   • Positive Control : Use a colony that will yield a product with this primers.