Team:Kyoto/Protocols

Making Media

For LB Media:

1. Wash a graduated cylinder with MilliQ.
2. Add the following to about 180ml of MilliQ in the graduated cylinder:

   Bacto-yeast extract

   1.0g (0.5w/v%)

   Tryptone

   2.0g (1.0w/v%)

   NaCl

   2.0g (1.0w/v%)

   1N NaOH

   200µl

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 (1.2w/v%) 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:

    Ampicillin

    100µg/ml

    Kanamycin

    50µg/ml

14. (Add 0.5 - 1.0w/v% Glucose for protein expression.)

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.

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

Phosphorylation of Primer

1. Mix the following.
   • 14µl : Primer (over 50pmole/µl [50µM])
   • 2µl : 10xProtruding End Kinase Buffer
   • 2µl : 10mM rATP (Dilluted "Code No:ATP-111")
   • 2µl : T4 Polynucleotide Kinase
2. Let stand for 1h at 37℃.
3. For 5min at 95℃ (Deactivate T4 Polynucleotide Kinase).
4. Store in the freezer.
5. Concentration of the phoshorylated primers is estimated at 10pmole/µl [10µM])

Standard 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.

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.

Mutagenesis

• (Point mutation, Deletion, Insertion)

1. Mix the following.

   10xBuffer

   5µl

   2mmol/l dNTP

   5µl

   Primer Forward (10pmol/µl)

   1.5µl

   Primer Reverse (10pmol/µl)

   1.5µl

   Template Plasmid (50ng/µl)

   1µl

   KOD plus ver.2

   1µl

   MilliQ

   35µl

   Total

   50µl

   • Control: Instead of KOD plus ver.2, add 1µl MilliQ.
2. PCR
   1. Let stand for 2min at 94℃.
   2. Xcycles (1cycle for 1kb) for 10s at 98℃ and for Ymin (1min for 1kb) at 68℃.
   3. Hold at 4℃.
3. Restriction Digests (DpnI)
   1. Take 25µl of the solutions into fresh tubes.
   2. Add 1µl DpnI (10U/µl).
   3. Let stand for 1h at 37℃.
4. Agarose gel electrophoresis, using 5µl of the solution for confirmation.
5. Ligation
   1. Mix the following.

      Sample

      2µl

      Ligation high

      5µl

      T4 Polynucleotide Kinase (5U/µl)

      1µl

      MilliQ

      7µl

      Total

      15µl

   2. Let stand for 1h at 16℃.
6. Transformation, using 10µl of the solution.

Gel Electrophoresis

1. Prepare 200ml of a 1.0% agarose solution:
   1. Measure 2.0g agarose into a beaker.
   2. Add 200ml 1xTAE buffer.
   3. Wrap the top of the beaker with plastic wrap.
   4. Punch a hole through the wrap with a pipette tip (To let out steam).
   5. Dissolve the agarose by heating in microwave and swirling without boiling.
   6. Allow the agarose to cool.
   7. Pour the agarose solution into a gel tray on a gel maker.
   8. If there is air bubbles, pushing them with a pipette tip.
   9. Place comb in the maker.
   10. Cover the maker with a plastic wrap.
   11. Let stand for about 45min.
   12. Remove the comb carefully.
   13. Store in the Tupperware in the refrigerator.
2. Place the tray in electrophoresis chamber.
3. Cover the tray with 1xTAE buffer.
4. Prepare samples for electrophoresi:
   1. Add 1µl of 6x Loading Buffer for every 5µl of DNA solution.
   2. Mix well.
5. Load 6µl of the DNA solution per well.
6. Electrophorese at 100volts for about 30min until Loading Buffer have migrated approximately three-quaters of the gel.
7. Stain the gel in 0.5µg/ml ethidium bromide for 20-30min.
8. Rinse the gel with MilliQ.
9. Place a plastic wrap on the transilluminator in the cabinet of Printgraph.
10. Place the gel on the transilluminator.
11. Turn on the transilluminator and confirm the position of the gel.
12. Shoot the picture.
13. Turn off the transilluminator.
14. Dispose of the gel.

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.

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,