Editing PCR Screening and Restriction Digestion (section)

== Restriction Enzyme Digestion ==

1. For PCR positive clones, transfer 10 ul culture to 5 ml LB/amp in 15 ml tube

2. Culture at 37 oC o/N in a shaker-incubator.

3. Spin down cells,

4, Add &nbsp;250 ul P1, suspend cells by votexing for 1~2 min.

5. Add 250 ul P2. Mix gentely, let lysis for 1 min

6. Add 350 ul N3. Mix&nbsp;

7. Transfer to 1.5 ml tube.

8, Spin 2 X 3 min.

9. Transfer supernatant to a miniprep column (column from China must be pretreated: soaked in 5 N NaOH for 30min, spin to remove NaOH from column)

10. Spin for 3 sec

11. Add 350 ul PB, spin, remove liquid

12. Add 700 ul PE, spin, remove liquid

13. Spin for 1 min at max speed

14. Add 50 ul EB or TE buffer, soak for 3 sec,&nbsp;

15. Spin for 1 min at max speed

16. Check DNA quality and quantitate DNA concentration using nanodrop

'''Digestion'''

Use 5 ul miniprep for &nbsp;digestion

1. make mix, per reaction

1.5 ul FastDigestion Buffer

0.3 ul enzyme 1

0.3 ul enzyme 2 (if required)

5 ul miniprep

H2O to 15 ul

2. Incubate at 37 oC &nbsp;for 30 min in an incubator&nbsp;

3. Add 5 ul 6X GRGreen loading buffer

4. Run on 1% agarose gel

5. Take a picture using blue light transilluminator&nbsp;

6. Process gel picture and upload to ELN

7. Document all experimental details