Team:KAIST Korea/Notebook Labnote

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.