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 I15008_B0034

grow overnight

|}