User:Hana Benzeer/Notebook/SGM Summer Project/2011/07/04

Purification (miniprep) of 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

Digestion of I15009 with XbaI/PstI
To digest 35μL of purified plasmid I15009:
 * 1) Add 0.5μL of 100x BSA
 * 2) Add 5μL of NEB 2
 * 3) Add 1μL of XbaI
 * 4) Add 1μL of PstI
 * 5) Add 12.5 μL of DNase free water
 * 6) Mix the sample then do quick spin.
 * 7) Place the tube in the 37°C incubator for 2 hrs.

Gel Preparation
Prepare 2% agarose gelP: 65 ml of 2% agarose gel:
 * 1) Weigh 1.3 g of agarose powder then transfer to flask
 * 2) Add 65 ml of 1x TAE buffer
 * 3) Mix and heat until no speckles remain
 * 4) Place on bench until cool
 * 5) Add 2μL of Ethidium Bromide. Swirl to mix
 * 6) Pour into prepare casting tray with comb and ensure no bubbles.
 * 7) Wait for 30-40 min for gel to set

2% gel, loading
Add 10μL of 6x loading buffer (LB) into each 50μL digested sample
 * 1) 10μL, 100bp NEB quick ladder
 * 2) Blank
 * 3) 50μL,I15009
 * 4) 5μL, I15009

The band 175 bp is cut from the gel and transferred into labelled eppendorf tube in which it is stored in the freezer at 21°C. The Gel is captured pic.

Transformation of I15010 with DH5-α
The I15010 is transformed with competent cell DH5-α.
 * 1) Fill up the box with ice.
 * 2) Take 1 eppendorf tube,DH5-α from the -80°C freezer and put it on ice.
 * 3) The I15009 is placed in the ice.
 * 4) Take out the Soc from the -20°C freezer°C and leave in the incubater for 10 min
 * 5) Transfer the I15009 into eppendorf. Mix the DNA to the cells by vortex and leave in the ice for 5 min. Note= while doing this, flame should be on.
 * 6) The eppendord tube is placed in 42°C waterbath for 1 min exactly
 * 7) Add 250μL of Soc.
 * 8) The tube is then placed in the shaker for 1 hr
 * 9) Take 1 selective agar plate, Ampicillin (Why? Because the and leave it in the incubater upside down with lid open slightly to lit air through.
 * 10) Take out the tubes from the shaker
 * 11) Take out the plates from the incubater.
 * 12) Turn on the flame. Transfer the contents from each epppendorf tubes into each 4 plates.
 * 13) Add glass beads.
 * 14) Now take all 4 plates and shake for 8-20 sec
 * 15) Remove the glass beads
 * 16) The plates are then left overnight in incubater at 37°C.

Inoculation of I15010 Inoculation of I15008_B0034


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