Materials

Procedure

1. Add 25g LB broth and 15g agar into 1L DDW.
2. Autoclave

Materials

Procedure

Pellet Cells

1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.
2. Centrifuge at 13000 rpm for 2.5minutes to pellet the cells.
3. Remove the supernatant.

Lyse Cells

4. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.
5. Centrifuge at 13000 rpm for 2.5minutes to pellet the cells.
6. Remove the supernatant.

Protein Precipitation

7. Add 200ul of Protein Precipitation Solution to the RNase-treated cell lysate. Vortex vigorously at high speed for 20seconds to mix the Protein Precipitation Solution tieh the cell lysate.
8. Incubate the sample on ice for 5minutes.
9. Centrifuge at 13,000rpm 3minutes.
10. Transfer the supernatant containing the DNA to a clean 1.5ml microcentrifuge tube.
Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in the tube to avoid contaminating the DNA solution with the precipitated protein.
11. Add 600ul isopropanol.
12. Gently mix by inversion until the thread-like strands of DNA form a visible mass.
13. Centrifuge at 13000rpm for 2minutes.
14. Carefully pour off the supernatant.
15. Add 600ul of room temperature 70% ethanol and gently invert the tube 5 times to wash the DNA pellet.
16. Centrifuge at 13000rpm for 2minutes. Carefully aspirate the ethanol.
17. Drain the tube on clean absorbent paper and allow the pellet to air-dry for 10-14minutes.
18. Add 50~100 μl TE buffer and rehydrate the DNA by incubating at 65℃ for 20min. Periodically mix the solution by gently tapping the tube. Alternatively, rehydrate the DNA by incubationg the solution overnight at room temperature or at 4℃.

Procedure

1) Restriction enzyme digestion

1. Incubate 2hr at 37℃.
2. Inactivation 20min at 65℃.

2) Dephosphorylation (Only for vector)

1. Incubate 30min at 37℃.
2. Inactivation 5min at 70℃.

3) Ligation

1. Incubate 16hr at 16℃.

4) Transformation

1. Add 20ul ligated vector into 100ul of competent cell.
2. Incubate ice 5min.
3. Heat shock 42℃, 1min 30sec.
4. Ice 5min.
5. Recovery with 700ul LB at 37℃, 1hr.
6. Plating.

Procedure

Reaction Conditions

1. 95℃ 3min
2. 95℃ 30sec
3. 54℃ 30sec
4. 72℃ 1min/kbp
5. 72℃ 10min
6. 12℃ ∞

Materials

• MGTM Gel Extraction SV - Macrogen

Procedure

1. Excise the DNA band of interest using an ethanol-cleaned razor blade.
2. Weigh the gel slice in a microcentrifuge tube. Add 3 volumes (ul) of Buffer GB to 1 volume (mg) of gel.
3. Incubate at 50°C until the agarose gel is completely melted (5 ~ 10 min).
4. Transfer the mixture to a MGTM SV column. Centrifuge for 1 min. Discard the pass-through and re-insert the MGTM SV column into the Collection Tube.
5. Add 700 ul of Buffer NW to the MGTM SV column. Centrifuge for 30 sec. Discard the pass-through. Reinsert the MGTM SV column into the Collection Tube.
6. Centrifuge for additional 1 min to remove residual wash buffer. Transfer the MGTM SV column to a new 1.5 ml tube.
7. Apply 30ul of Buffer EB to the center of the membrane in the MGTM SV column, let stand for 1 min, and centrifuge for 1 min.

Materials

• AccuPrep® PCR Purification Kit - BIONEER

Procedure

1. Add 5 volumes of PB Buffer to 1 volume of the PCR reaction.
2. Apply the sample to the Binding column tube to bind DNA.
3. Centrifuge for 30-60 sec to make the sample pass through the Binding column tube.
4. Discard flow-through and place the Binding column tube in the same tube.
5. Add 500μl of WB Buffer to the Binding column tube and centrifuge for 30-60 sec to wash.
6. Discard flow-through and place the Binding column tube in the same tube again.
7. Centrifuge the Binding column tube for an additional 1min for drying.
8. Place the Binding column tube in a clean 1.5 ml tube.
9. Add 30μl of EL Buffer to the center of the Binding column filter, and let the column stand for 1 min.
10. Centrifuge for 1 min to elute.

Materials

• AccuPrep® plasmid mini extraction Kit – BIONEER

• Buffer 1 - Resuspension buffer
• Buffer 2 - Lysis buffer
• Buffer 3 - Neutralization buffer
• Buffer 4 - Washing buffer
• Buffer 5 - Elution buffer

Procedure

1. Add 250ul of Buffer 1 to the collected cells and completely resuspend by vortexing or pipetting.
2. Add 250ul of Buffer 2 and mix by inverting the tube 3-4 times gently.
3. Add 350ul of Buffer 3 and immediately mix by inverting the tube 3-4 times gently.
4. Centrifugation the tube at 13000rpm, 4℃ for 10min.
5. Transfer the cleared lysate to the DNA binding column tube and centrifuge 1min pour off the flow-through and re-assemble the DNA binding filter column with the 2mL collection tube.
6. Add 700ul of Buffer 4 to the DNA binding column tube and centrifuge 1min. Pour off the flow-through and re-assemble the DNA binding filter column with the 2mL collection tube.
7. Dry by additional centrifuge 1min.
8. Transfer the DNA binding filter column to the new 1.5mL EP tube.
9. Add 30ul of buffer 5 to the DNA binding filter column and wait 1min.
10. Elute the plasmid DNA by centrifuge 1min.

Materials

Procedure

1) Pre-culture

1. Inoculate MG1655(containing pKD46) into 3mL LB broth(1% Ap) at 30℃.
2. Grow overnight.

2) DNA Preparation

1. PCR Km cassette from pKD13

( Reaction conditions )
• (1) 95℃ 3min
• (2) 95℃ 30sec
• (3) 54℃ 30sec
• (4) 72℃ 1min 30sec
• (5) 72℃ 10min
• (6) 12℃ ∞

2. Check product with gel electrophoresis and purify appropriate method. (PCR purification or Gel extraction)

3) Cell culture

1. Inoculate 3mL pre-cultured cells into 50mL of LB (1% Ap). Set OD = 0.03.
2. Incubate 30℃.
3. When OD = 0.1, add 500uL of arabinose.
4. Check OD every hour.

4) Washing

※ Every single step in washing must be done in ice.

1. When OD = 0.5 ~ 0.7, transfer cells into 50mL falcon tube.
2. Centrifuge for 15min at 3000rpm, 4℃. (Pre-chill centrifuge)
3. Discard supernatants.
4. Resuspend pellet with 1mL of glycerol. (10%, pre-chilled pipet tip)
5. Add 49mL of glycerol(10%). Shake by inverting 5 times.
6. Repeat 8 – 10 two more round.
7. Resuspend pellet & transfer into ep-tube.

5) Electro-transformation

※ Every single step from 1 to 7 must be done in ice.

1. Mix ≒1000ng of insert DNA with washed cells.
2. Incubate in ice for 30min.
3. Transfer 120uL of mixture into electroporation cuvette.
4. Shock once.
5. Recover in ice for 5min.
6. Resuspend cells with 1mL of LB.
7. Transfer 1mL of cells into new ep-tube.
8. Recover in 30℃ shaking incubator for 1hr.
9. Spread onto Km plate. (30min pre-warmed plate)
10. Incubate overnight at 30℃.