User:Hana Benzeer/Notebook/SGM Summer Project/2011/06/30

Check overnight growth (4x100ml)

Part	strain	Growth
BBa_I15010	DH5-α	No
BBa_I15010	DH5-α	No
BBa_I15008	DH5-α	Yes
BBa_I15008	DH5-α	Yes

1. The tubes I15010 are left in the shaker overnight.
2. Take two tubes, BBa_I15008, DH5-α.
3. Remove 700μL for each tube and transfer into eppendorf tibe.
4. Add 300μL of 50% glycerol stock into each eppendorf tube.
5. Mix by vortex.
6. Store in -21°C freezer.
7. Spin the 10ml tubes for 10 min at 70.
8. Remove the supernatant
9. Store the tubes in -21°C freezer.

Purification of B0034, I15008 and I15009

10 ml of overnight growth was spin down and supernatant discarded.

1. Add 500μL of P1 resuspension buffer. resuspend using the pipette until all cell pellets are liquified
2. labelled 2 eppendorf tubes
3. Transfer 250μL of the resuspended cells into each eppendorf tubes.
4. For each tubes, add 250μL of P2 lysis buffer.
5. Wait for 3-4 min for lysis to occur.
6. For each tubes, add 350μL of N3 neutralisation buffer to stop the lysis.
7. Invert the tubes 10 times until the percipitation occurs.
8. Centriguge for 10 min at 13000 rpm
9. Decant the supernatant in each of two correspondingy labelled QIAgen quick columns, ensure no percipitate enters the column
10. Centrifuge 1 min at 13000 rpm to bind the DNA to the column. Discard the flow-through.
11. Add 500μL of PB wash buffer into each columns.
12. Centrifuge for 1 min at 13000 rmp. Discard the flow-through.
13. Add 750μL of PE final wash with ethanol into each column.
14. Centrifuge for 1 min at 13000 rmp. Discard the flow-through.
15. IMPORTANT! To remove any ethanol residues. Centrifuge for 1 additional min at 13000 rmp. Discard the flow-through.
16. place each column into new labelled eppendorf tubes.
17. Add 35μL of DNase free water to the centre of the column.
18. Wait for 1 min.
19. To elute, centrifuge for 1 min at 13000 rmp.
20. Place the sample back on ice or store at -20°C