Haynes Lab:Notebook/Engineering PC-TFs/2014/11/05

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Summary

  • Gel Extracted/Purified BL01
  • Retransformed BL01 & BL05
  • Digested CMV/MV9 with XbaI for LCR
  • LCR
  • Digested CMV/MV9 with XbaI and desphos'd, clean and concentrated for long transformation
  • Long Transformation

Gel Extraction/Purification of BL01

Reagent Volume Expected:
1. BL01 = 2500bp
DNA(BL01) 15.0 μL
10X buffer 3.0 μL
XbaI 1.0 μL
SpeI 1.0 μL
dH2O 10 μL
Total 30 μL --> 37°C/ 15 min.



Gel Purification: BL01/V0120

  • Using Prior Digest
  • Placed 15μL 1kB Ladder in well one and 30μL of BL01 digest in well two.
  • Ran gel at 110V for 55 minutes.
  • Placed gel on UV transilluminator and cut out desired band(2500bp).
  • Used Zymo gel purification kit.
  • Assumed 200mg gel, added 600μL ADB to the 1.5mL tube containing extracted gel piece.
  • Put on heat block at 55°C for 8 minutes.
  • Put ~750μL gel/ADB into spin column, centrifuged at max rpm for 30 seconds.
  • Pipetted 200μL wash buffer to the tube, centrifuged at max rpm for 30 seconds. Repeated once.
  • Transferred column to new labeled 1.5mL tube.
  • Pipetted 15μL elution buffer to the column.
  • Spun at max rpm for 30 seconds.

Concentration

Plasmid OD260 OD260/280 ng/μL
1. Purified BL01 0.03 2.05 30.272


Digest and Dephos of CMV/MV9 with XbaI

DNA(CMV/MV9) Concentration: 163ng/μL 5.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
dH2O 7.5 μL
Total 15 μL --> 37°C/ 15 min.


  • Clean and concentrated XbaI cut CM9. Eluted with 10μL dH2O.
Mixture from Above Digest 10.0 μL
10X Phosphotase buffer 2.0 μL
Phosphotase 1.0 μL
dH2O 7.0 μL
Total 20 μL --> 37°C/ 10 min. 75°C for 2 min.


  • Clean and concentrated DNA after dephos step; eluted with 12μL dH2O.


Plasmid OD260 OD260/280 ng/μL
1. Purified DNA 0.017 1.953 17.425

LCR
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
  • 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.


















Normal Transformation (BL01 & BL05 in V0120)

  • 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.



Long Transformation
Using the set up ligation products from above.

  • DH5α-T cells were thawed in ice.
  • Two 1.5mL tubes were setup and labeled; LCR BL01 & Long Transformation BL01.
  • 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.



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