Haynes Lab:Notebook/Engineering PC-TFs/2014/08/04

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Ligation Calculations resulted in the following:

  Ligation Negative Control
Insert DNA (BL01, BL05, BL09 respectively) 12μL none
Vector DNA (50 ng) 1μL same
10x Biolabs T4 Ligase Buffer 2.0 μl same
Biolabs T4 Ligase 1.0 μl same
dH2O 4.0μL 16.0μL
  20.0 μL total    same

Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 45 minutes.

Long Transformation
Using the set up ligation products from above.

  • DH5α-T cells were thawed in ice.
  • Six 1.5mL tubes were setup and labeled; 1 (BL01), 5 (BL05), 9 (BL09), CMV9 (CMV/MV9, ligation benchmark), rhlI (pos control), Neg (negative control).
  • 60μL of thawed cells were mixed and then transferred into each of these six empty tubes.
  • The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice.
  • The RhlI plasmid was used as a positive control; 1μL of this plasmid was added to 19μL dH2O to equal the other two products' 20μL total volume. Added to the fifth tube with 60μL thawed cells.
  • All tubes incubated on ice for 35 minutes.
  • All tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes.
  • 900μL LB broth (no antibiotic) was pipetted into each of the three tubes.
  • Tubes were taped into an empty plate. The plate was taped to the shaker within the incubator.
  • Incubated on shaker for 1 hour at 37°C and 240rpm.
  • Centrifuged for 1.5 minutes at 8 x g.
  • 500μL media removed from each tube.
  • Resuspended cell pellet in remaining 400μL media.
  • 300μL cells transferred onto respective plate.
  • Sterile glass beads used to spread cells onto plate.
  • Placed in the incubator for overnight growth.