Haynes Lab:Notebook/Engineering PC-TFs/2014/10/11

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Summary

  • Split Cells
  • Purified BL09 Negative Control
  • LCR

Purified BL09 Negative Control Protein Extract
Protein kept on ice during procedure; centrifuge in cold room used.

  • Vortexed His-Affinity Gel briefly.
  • Transferred 250μL of His-Affinity Gel to the Zymo-Spin P1 column.
  • Placed column in 2mL collection tube.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded flow-through.
  • Added 300μL PcTF in protein extraction buffer to column.
  • End-over-end mixing for two minutes to resuspend gel and allow protein to bind to resin.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded flow-through.
  • Added 250μL His-Wash Buffer and mixed to resuspend gel.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded flow-through.
  • Repeated wash step once.
  • Transferred column into a labeled 1.7mL microcentrifuge tube.
  • Added 150μL His-Elution Buffer and resuspended gel via end-over-end mixing for one minute.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded column and stored purified protein at -20°C.

Digest

Reagent Volume
DNA(CMV/MV9) 8.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
dH2O 4.5μL
Total 15 μL --> 37°C/ 35 min.


Standard procedure was followed to clean and concentrate 8μL XbaI cut vector (eluted with 6μL H2O.

LCR
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).

Diluted insert and vector down to 3nM using MW and known concentration.

LCR Calculations

  • DNA Part to Oligo Bridge is 1:10 (DNA part ratio is 1:1)
  • X ng insert = (bp inset / bp vector) x 1 x 50 ng vector
  • X ng BL01&9 = (2520bp/5197bp)*1*50ng vector = 24.24ng BL01&9
  • Volume BL01&9 = 24.24ng*(1μL/10ng) = 2.424μL
  • Volume CMV/MV9 = 50ng*(1μL/69ng) = 0.72μL


  BL01 BL05
Insert DNA 3μL 6μL
XbaI Cut and Cleaned Vector DNA (CMV/MV9) 1μL 1μL
10X Ampligase Buffer 2.5 μl 2.5μL
Ampligase 0.5 μl 0.5μL
Betaine 2.2μL 0μL
dH2O 13.3μL 12.5μL
Total 25.0 μL    25μL
Mix the reaction(s) thoroughly by flicking the tube.
Placed in thermocycler on LCR setting.















Transformation

  1. Warmed selection agar plates at 37°C.
  2. Incubated DH5α Turbo competent cells on ice just until thawed. Use 50 μL per transformation.
  3. Added 50 μL thawed cells to 5μL LCR product. Immediately placed on ice and incubated for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance)
  4. Pipetted the total volume of cells + LCR reaction onto the agar; spread using sterile glass beads.
  5. Incubated overnight at 37°C.