ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry
LCR Assembly
Prep the Bridge Oligos
Note: Final conc. in LCR rxn. is 30 nM each
- 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
- 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
|