ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry
LCR Assembly
Prep the Bridge Oligos
Note: Final conc. in LCR rxn. will end up being 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) in a final volume of 50 μL
- Calculation: The volume of purified dsDNA (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
Set up the following in PCR tubes:
| 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
|
- Thermal cycler: program the following...
- Incubate at 37°C/ 30 min.
- Heat-inactivate PNK at 65°C/ 20 min.
LCR Reaction
- Using the entire PNK DNA reaction, add other components (into the same PCR tubes) as described in the table below
- Best way to do this is to make a master mix, then aliquot into PNK DNA
Master Mix (in 1.5 mL tube)
| Reagent |
Volume
|
| Oligo Bridge 1 |
2.0 (each)
|
| 10X Ampligase Buffer |
2.0
|
| Ampligase |
1.0
|
| dH2O |
3.0 μL
|
| |
20.0 μL
|
Add master mix into 10 μL PNK DNA
| Reagent |
Volume
|
| PNK DNA |
10.0 μL
- Run LCR...
- Look for "LCR" program on one of the PCR machines
Transformation
- Carry out the usual transformation. (quick and dirty.)
|
|
Open Chromatin
