| 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.
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.
- 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.
- Warmed selection agar plates at 37°C.
- Incubated DH5α Turbo competent cells on ice just until thawed. Use 50 μL per transformation.
- 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)
- Pipetted the total volume of cells + LCR reaction onto the agar; spread using sterile glass beads.
- Incubated overnight at 37°C.