Haynes Lab:Notebook/Engineering PC-TFs/2015/03/23

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  • LCR
  • Met Kodibagkar grad student Shubanghi

Oligo bridges initially 30nm pellet. Diluted down to 30nM in 25μL reaction volume (Added 300μL H2O to IDT tube with pelleted DNA to yield 100μM, added 3μL of that dilution to 97μL H2O to yield 3μM, added 5μL of that dilution to 45μL H2O to yield 300nM, added 2.5μL of this dilution to the 25μL reaction volume for LCR).

Dephos XbaI linearized MV10 gel purification product

XbaI gel purified MV10 5.0 μL
10X Phosphotase buffer 2.0 μL
Phosphotase 1.0 μL
dH2O 12.0 μL
Total 20 μL --> 37°C/ 10 min. 75°C for 2 min.

LCR Calculations

  • 1:1 vector to insert
  • BL01: (2520bp/5197bp)(1)(50ng)=24.24ng
  • 24.24ng(1μL/11.3ng)=2.145μL XbaI/SpeI cut BL05 Sample2
  • BL05: (2520bp/5197bp)(1)(50ng)=24.24ng
  • 24.24ng(1μL/18.5ng)=1.31μL XbaI/SpeI cut BL05 Sample1
  • 50ngCMV/MV9(1μL/59ng)=0.847μL XbaI cut dephos'd CMV/MV9
  • 50ngCMV/MV9(1μL/15ng)=3.33μL XbaI cut dephos'd CMV/MV9

  BL05.1 BL05.2 Neg
Insert DNA 1.3μL 2.2μL 0μL
XbaI Cut Dephos'd CMV/MV9 1μL 3.33μL 0μL
Oligo Bridge1 2.5μL 2.5μL 2.5μL
Olido Bridge2 2.5μL 2.5μL 2.5μL
10X Ampligase Buffer 2.5 μl 2.5μL 2.5μL
Ampligase 1μl 1μL 1μL
Betaine 0μL 0μL 0μL
DMSO 0μL 0μL 0μL
dH2O 14.2μL 11μL 14.5μL
Total 25.0 μL    25μL
Mix the reaction(s) thoroughly by flicking the tube.
Placed in thermocycler on LCR setting. *Volume reduced to ~10μL after removal from PCR machine. Settings incorrect?

Long Transformation
Using the set up ligation products from above.

  • Warmed selection agar plates at 37°C.
  • DH5α-T cells were thawed in ice.
  • 1.5mL tubes were setup and labeled.
  • 60μL of thawed cells were mixed and then transferred into each of these two empty tubes.
  • The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice.
  • 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 SOC Medium 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 9 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 at 37°C.