User:Hana Benzeer/Notebook/SGM Summer Project/2011/07/01

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Digestion of purified plasmid

Take out 3 eppendorf tubes, B0034, I15008 and I15009 from the -21°C freezer

Digestion of B0034 with PstI/SpeI

To digest 35μL of purified plasmid B0034:

  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 I15008 with PstI/XbaI

To digest 35μL of purified plasmid I15008:

  1. Add 0.5μL of 100x BSA
  2. Add 5μL of NEB#2
  3. Add 1μL of SpeI
  4. Add 1μL of XbaI
  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 I15009 with PstI and XbaI

To digest 35μL of purified plasmid I15009:

  1. Add 0.5μL of 100x BSA
  2. Add 5μL of NEB#2
  3. Add 1μL of SpeI
  4. Add 1μL of XbaI
  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. Blank
  3. 50μL, B0034 (expected, ~21 bp)
  4. 5μL, B0034(expected, ~21 bp)


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,I15008
  4. 5μL, I15008
  5. Blank
  6. 50μL,I15009
  7. 5μL, I15009

The band is cut from the gel. The bands that cutted are B0034, I15008 and I15008. Then the pic of the gel is taken. pic.

Gel, B0034, I15008 and I15009 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 30μL of free nuclease water to the centre white filter column.
  10. Centrifuge for 1 min at 13000 rpm.
  11. The column is removed from the eppendorf tube
  12. The eppendorf tubes are placed in the ice or stored in the -21°C freezer.

Ligation

Ligation of ribosome binding site B0034 to I15008 and I15009. Ligating between the B0034 to I15008:

    1. Add 1μL of ligase
    2. Add 1μL of ligase buffer
    3. Add 1μL of backbone, B0034
    4. Add 7μL of insert, I15008

Ligating between the B0034 to I15009: Probelm with the sample as the gel is not dissolved very well. This need to be repeated on the 4th of July.

Transformation of I15008_B0034

The I15008_B0034 are 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 first ligated I15008_B0034 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 I15008_B0034 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



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