User:Hana Benzeer/Notebook/SGM Summer Project/2011/06/30
From OpenWetWare
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 |
- The tubes I15010 are left in the shaker overnight.
- Take two tubes, BBa_I15008, DH5-α.
- Remove 700μL for each tube and transfer into eppendorf tibe.
- Add 300μL of 50% glycerol stock into each eppendorf tube.
- Mix by vortex.
- Store in -21°C freezer.
- Spin the 10ml tubes for 10 min at 70.
- Remove the supernatant
- Store the tubes in -21°C freezer.
Purification of B0034, I15008 and I15009
10 ml of overnight growth was spin down and supernatant discarded.
- Add 500μL of P1 resuspension buffer. resuspend using the pipette until all cell pellets are liquified
- labelled 2 eppendorf tubes
- Transfer 250μL of the resuspended cells into each eppendorf tubes.
- For each tubes, add 250μL of P2 lysis buffer.
- Wait for 3-4 min for lysis to occur.
- For each tubes, add 350μL of N3 neutralisation buffer to stop the lysis.
- Invert the tubes 10 times until the percipitation occurs.
- Centriguge for 10 min at 13000 rpm
- Decant the supernatant in each of two correspondingy labelled QIAgen quick columns, ensure no percipitate enters the column
- Centrifuge 1 min at 13000 rpm to bind the DNA to the column. Discard the flow-through.
- Add 500μL of PB wash buffer into each columns.
- Centrifuge for 1 min at 13000 rmp. Discard the flow-through.
- Add 750μL of PE final wash with ethanol into each column.
- Centrifuge for 1 min at 13000 rmp. Discard the flow-through.
- IMPORTANT! To remove any ethanol residues. Centrifuge for 1 additional min at 13000 rmp. Discard the flow-through.
- place each column into new labelled eppendorf tubes.
- Add 35μL of DNase free water to the centre of the column.
- Wait for 1 min.
- To elute, centrifuge for 1 min at 13000 rmp.
- Place the sample back on ice or store at -20°C