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Miniprep of cells

  • The pSB1C3 RFP cells that were incubated in luria broth overnight yesterday, were minipreped using the OMEGA bio-tek protocol, in order to isolate plasmid of interest.

Nanodrop results

SampleVolumeConcentration ng/ul260/280260/230

Double Digest of the pSB1C3 miniprep

Samples 1 and 2 were transfered to new eppendorf tubes

  • In each tube it was added
    • 39ul of DNA (miniprep)
    • 1ul of XbaI
    • 2ul of SpeI
    • 6ul of cut smart buffer
    • 12ul of sterile water

The samples were incubated at 37 degrees water buffer After the samples were incubated at 80 degrees to kill the enzymes

Transformation of TOD genes ligations done on the 02/09/13 using the pGEM-T Vector System I-A3600 (changes were made to the protocol)

  • Centrifuge the ligations reactions briefly.
  • Add 5ul of each ligation reaction to a sterile 1.5ml
  • Thaw the competent cells on ice. Mix the cells by gently flicking the tube.
  • Carefully transfer 100ul of the competent cells to the ligation reaction tubes.
  • Incubate the tubes on ice for 20 minutes
  • Heat-shock the cells for 45-50 seconds in a water bath at exactly 42C degrees. Do not Shake. Immediately return the tubes to ice for 2 minutes.
  • Add 950ul of room temperature SOC medium to the ligation reaction transformations. incubate for 1.5 hours at 37C with shaking.

Plating out the transformants

  • The transformants would be plated out on Ampicillin resistant plates that were prepared yesterday.
  • 40ul of X-Gal and 20ul of of IPTG were streaked respectively unto the already made plates. (except the viability and resistance test plates)
Below  is a list of the plates that were made;

1)x2 plates of positive control (no PCR product)- 20ul and 200ul plates 2)x2 plates of Background control- 20ul and 200ul 3)x2 plates of ligated product 1 (L1) - A- 20ul and 200ul plates 4)x2 plates of L2- C -20ul and 200ul plates 5)x2 plates of L3- E - 20ul and 200ul plates 6)x2 transformation control - (50pg of puc19) - 20ul and 200ul plates 7)x1 Viability control (only luria broth)- 200ul of cells 8)x1 resistance control (Only Amp agar) - 200ul of cells

All 14 plates were incubated overnight at 37 degrees centigrade.