User:Hana Benzeer/Notebook/SGM Summer Project/2011/07/04
From OpenWetWare
Purification (miniprep) of 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
Digestion of I15009 with XbaI/PstI
To digest 35μL of purified plasmid I15009:
- Add 0.5μL of 100x BSA
- Add 5μL of NEB 2
- Add 1μL of XbaI
- Add 1μL of PstI
- 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 2% agarose gelP: 65 ml of 2% agarose gel:
- Weigh 1.3 g of agarose powder then transfer to flask
- Add 65 ml of 1x TAE buffer
- Mix and heat until no speckles remain
- Place on bench until cool
- Add 2μL of Ethidium Bromide. Swirl to mix
- Pour into prepare casting tray with comb and ensure no bubbles.
- Wait for 30-40 min for gel to set
2% gel, loading
Add 10μL of 6x loading buffer (LB) into each 50μL digested sample
- 10μL, 100bp NEB quick ladder
- Blank
- 50μL,I15009
- 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.
- Fill up the box with ice.
- Take 1 eppendorf tube,DH5-α from the -80°C freezer and put it on ice.
- The I15009 is placed in the ice.
- Take out the Soc from the -20°C freezer°C and leave in the incubater for 10 min
- 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.
- The eppendord tube is placed in 42°C waterbath for 1 min exactly
- Add 250μL of Soc.
- The tube is then placed in the shaker for 1 hr
- 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.
- Take out the tubes from the shaker
- Take out the plates from the incubater.
- Turn on the flame. Transfer the contents from each epppendorf tubes into each 4 plates.
- Add glass beads.
- Now take all 4 plates and shake for 8-20 sec
- Remove the glass beads
- The plates are then left overnight in incubater at 37°C.
Inoculation of I15010
Inoculation of I15008_B0034
grow overnight
|}