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

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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.

Purification of B0034

Purification of I15009

Digestion of B0034

Digestion of I15009

pepare the gel 2%

run the gel

Phsically cut the gel of particular bands

Gel purification

Ligation of B0034 with I15009

    1. Add 7 μL of B0034
    2. Add 1μL of I15009
    3. Add 1μL of T4 ligase buffer
    4. Add 1μL of T4 ligase Total= 10μL

Transformation of ligated B0034_I15009

    1. Add 10μL of Ligated B0034_I15009 into competent cell, DH5α