User:Hana Benzeer/Notebook/SGM Summer Project/2011/06/27

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Transformation of light sensor parts

The light sensor part consistutes of:

  1. BBa_B0034, Ampicillin resistence
  2. BBa_B0015, Ampicillin/Kanamycin resistence
  3. BBa_I15010, Kanamycin resistence
  4. BBa_I15009,Kanamycin resistence
  5. BBa_I15008,Kanamycin resistence
  6. BBa_R0040,Ampicillin resistence
  7. BBa_I13453,Ampicillin resistence

The plasmids are transformed with competent cell, strain XL-1-Blue.

  1. Fill up the box with ice.
  2. Take 7 eppendorf tubes containing competent cells, XL-1-Blue from the -80°C freezer and straight into ice.
  3. Take the plasmids (Igem plates 2010. If the plates are not used, meaning the well of interest is not puncrures, add 10μL of free nuclease water)from the -20°C freezer
  4. Take out the Soc from the -20°C freezer°C and leave in the incubater for 10 min
  5. Add 2μL from each plasmid and transfer into each 7 competent cells. 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. Take 7 eppendorf tubes into 42°C waterbath and leave it for 1 min exactly
  7. Add 250μL of Soc into each 7 eppendorf tubes.
  8. The tubes are then left in the shaker for 1 hr
  9. Take 7 selective agar plates 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 7 plates.
  13. Add glass beads.
  14. Now take all 7 plates and shake for 8-20 sec
  15. Remove the glass beads
  16. The plates are then left overnight in incubater at 37°C

Results: show pic

Making Soc

Three flasks are made, flask 1 Sob, flask 2 Magnesium chloride, flask 3 D-glucose To make Sob:

    1. Add 5g of bactotypton
    2. Add 0.5g of sodium chloride
    3. Add 1g of bacto-yeast extract
    4. Add 0.0327g of potassium chloride
    5. Add 200 ml of distilled water
    6. Autoclave, standard media

To make Magnesium chloride:

    1. Add 0.2033 g of Mgcl2
    2. Add 100 ml of distilled water
    3. Autoclave

To make D-glucose:

    1. Add 0.182 g, D-glucose
    2. Add 100 ml of distilled water
    3. Autoclave, sugar run