User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/04: Difference between revisions

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Line 59: Line 59:
| Reagent || Volume
| Reagent || Volume
|-
|-
| PNK DNA || 10 μL
| PNK DNA || 10.0 μL
|-
|-
| Oligo Bridge || 3.0 (each)
| Oligo Bridge || 3.0 (each)
Line 67: Line 67:
| Ampligase || 1.0
| Ampligase || 1.0
|-
|-
| dH<sub>2</sub>O || x μL
| dH<sub>2</sub>O || 3.0 μL
|-
|-
| &nbsp; || 30.0 μL
| &nbsp; || 20.0 μL
|}
|}


Revision as of 22:28, 3 August 2015

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ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry

  • LCR Assembly


LCR Assembly

Prep the Bridge Oligos
Note: Final conc. in LCR rxn. is 30 nM each

  1. Make a 300 nM working solution (final volume = 100 μL) in a new tube. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL
  2. Use 3.0 μL of oligo working sln. per 30 μL LCR reaction


PNK treatment
Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups

  • Dilute the purified dsDNA to 30 fmol/μL (30 nM)
    • The volume of purified DNA (x) you will need to dilute in a final volume of 50 μL = length in bp ÷ measured ng/μL * 30 fmol/μL * 650 fg/fmol dsDNA ÷ 1,000,000 fg/ng * 50 μL final volume.
    • Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
  • Use 2.0 μL of each diluted dsDNA per 10 μL PNK reaction
  • Mix the appropriate amount of dsDNA fragments together and treat with Polynucleotide kinase to add 5'-phosphates (see table below)
Reagent Volume
30 fmol/μL MV10 2.0
30 fmol/μL Gal4DB-mCh 2.0
30 fmol/μL ATF2 2.0
10x T4 Ligation buf (NEB) 1.0
T4 PNK (NEB) 0.5
dH2O 2.5 μL
  10.0 μL
  • Incubate at 37°C/ 30 min.
  • Heat-inactivate PNK at 65°C/ 20 min.


LCR Reaction

  • Add components to the PNK DNA reaction as described in the table below
  • Set up the reactions in PCR-sized tubes
Reagent Volume
PNK DNA 10.0 μL
Oligo Bridge 3.0 (each)
10X Ampligase Buffer 3.0
Ampligase 1.0
dH2O 3.0 μL
  20.0 μL
  • Run LCR...
    • Look for "LCR" program on one of the PCR machines


Transformation

  • Carry out the usual transformation. (quick and dirty.)



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