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

Check overnight growth (4x100ml)
{|class="wikitable sortable" border="1" ! scope="col" | Part ! scope="col" | strain ! scope="col" | Growth {|
 * BBa_I15010 || DH5-α || No
 * BBa_I15010 || DH5-α || No
 * BBa_I15008 || DH5-α || Yes
 * BBa_I15008 || DH5-α|| Yes
 * BBa_I15008 || DH5-α || Yes
 * BBa_I15008 || DH5-α|| Yes
 * 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