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

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  • LCR
  • Long Transformation

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&5 = (2520bp/5197bp)*1*50ng vector = 24.24ng BL01&9
  • Volume BL01&5 = 24.24ng*(1μL/10ng) = 2.424μL
  • Volume CMV/MV9 = 50ng*(1μL/33ng) = 1.28μL
  • 2:1 vector to insert use 2.56μL vector.

  BL01 BL05
Insert DNA 2.5μL 2.5μL
XbaI Cut and Cleaned Vector DNA (CMV/MV9) 1.3μL 2.6μL
Oligo Bridge1 2.5μL 2.5μL
Olido Bridge2 2.5μL 2.5μL
10X Ampligase Buffer 2.5 μl 2.5μL
Ampligase 0.5μl 0.5μL
Betaine 0μL 0μL
DMSO 0μL 0μL
dH2O 13.2μL 11.9μL
Total 25.0 μL    25μL
Mix the reaction(s) thoroughly by flicking the tube.
Placed in thermocycler on LCR setting.

Long transformation protocol used for LCR reaction mixture.

Ligation Calculations resulted in the following:

  Ligation Negative Control
Insert DNA (BL01, BL05 respectively) 2.5 μL none
Vector DNA (50 ng) 1.3 μL same
10x Biolabs T4 Ligase Buffer 2.0 μl same
Biolabs T4 Ligase 1.0 μl same
dH2O 13.2 μL 15.7 μL
  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.
  • Clean 1.5mL tubes were setup and labeled.
  • 60μL of thawed cells were mixed and then transferred into each tube.
  • The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice.
  • BL05 in V0120 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.