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

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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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