User:Hana Benzeer/Notebook/SGM Summer Project/2011/06/20

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Purification of M30109
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 30μL 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.

Place the sample back on ice or store at -20°C

Digestion of M30109 with SpeI
To digest 30μL of purified plasmid M30109:
 * 1) Add 0.5μL of 100x BSA
 * 2) Add 5μL of NEB#2
 * 3) Add 1μL of SpeI
 * 4) Add 13.5 μL of DNase free water

Mix the sample then do quick spin. Place the tube in the 37°C incubator for 2 hrs

Digestion of M30109 with PstI
To digest 30μL of purified plasmid M30109:
 * 1) Add 0.5μL of 100x BSA
 * 2) Add 5μL of NEB#3
 * 3) Add 1μL of SpeI
 * 4) Add 13.5 μL of DNase free water

Mix the sample then do quick spin. Place the tube in the 37°C incubator for 2 hrs

Digestion of M30109 with PstI and SpeI
To digest 30μL of purified plasmid M30109:
 * 1) Add 0.5μL of 100x BSA
 * 2) Add 5μL of NEB#2
 * 3) Add 1μL of SpeI
 * 4) Add 1μL of PstI
 * 5) Add 12.5 μL of DNase free water

Mix the sample then do quick spin. Place the tube in the 37°C incubator for 2 hrs

Digestion of R0082_E0840 with PstI and XbaI
To digest 30μL of purified plasmid R0082_E0840:
 * 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

Mix the sample then do quick spin. Place the tube in the 37°C incubator for 2 hrs

Gel Preparation
Prepare 1% and 2% agarose gelP: For 65 ml of 1% agarose gel:
 * 1) Weigh 0.65 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

For 65ml 2& agarose gel. Follow recipe above but use double amount of agarose powder (1.3 g)

1% gel, loading
Add 10μL of 6x loading buffer (LB) into each 50μL digested sample
 * 1) 10μL, 1kb NEB quick ladder
 * 2) 50μL, M30109, SpeI (expected, ~7.3kb)
 * 3) 5μL, M30109, SpeI (expected, ~7.3kb)
 * 4) 50μL, M30109, PstI (expected, ~7.3kb)
 * 5) 5μL, M30109, PstI (expected, ~7.3kb)
 * 6) 50μL, M30109, PstI/SpeI (expected, ~7.3kb)
 * 7) 5μL, M30109, PstI/SpeI (expected, ~7.3kb)
 * 8) 10μL, 1Kb NEB quick ladder

2% gel, loading
Add 10μL of 6x loading buffer (LB) into each 50μL digested sample
 * 1) 10μL, 100bp NEB quick ladder
 * 2) 50μL, R0082_E0840,XbaI/PstI
 * 3) 5μL, R0082_E0840, XbaI/PstI
 * 4) 5μL, E0840#1, XbaI/PstI
 * 5) 5μL, E0840#2, XbaI/PstI
 * 6) Blank
 * 7) Blank
 * 8) Blank

Gel purification

 * 1) Add 300μL of QG solubilization buffer to the gel of the ependorf tube. Vortex.
 * 2) Place in the waterbath at 50°C for 10 min. Mix at intervals until the gel has completely dissolved.
 * 3) Add 100μL of isopropanol, mix. Transfer the contents into the quiagen quick gel column.
 * 4) Centrifuge for 1 min at 13000 rpm and discard the flow through.
 * 5) Add 500μL of QG buffer solubilisation buffer and centrifuge for 1 min at 13000 rpm. Discard the flow through
 * 6) Add 750μL of PE buffer that contains ethanol. Centrifuge for 1 min at 13000 rpm and discard the flow through.
 * 7) Centrifuge again to remove any ehtanol residues.
 * 8) Move the column into new labelled eppendorf tube.
 * 9) Add 30

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