Haynes Lab:Notebook/Engineering PC-TFs/2014/10/20

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Summary

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/33ng) = 1.28μL


  BL01 BL05
Insert DNA 3μL 3μL
XbaI Cut and Cleaned Vector DNA (CMV/MV9) 2μL 2μL
Oligo Bridge1 2.5μL 2.5μL
Olido Bridge2 2.5μL 2.5μL
10X Ampligase Buffer 2.5 μl 2.5μL
Ampligase 0.5 μl 0.5μL
Betaine 2.2μL 0μL
DMSO 2μL 0μL
dH2O 7.8μL 12.0μL
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.


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