ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry
LCR Assembly
Prep the Bridge Oligos
- MV10/Gal4DB bridge: LCRb_MV10tctGal4DB_rc
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 treatments
- MV10 + Gal4DB-mCh + ATF2
- MV10 (neg ctrl.)
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 |
Rxn1 |
Rxn2
|
30 fmol/μL MV10 |
2.0 |
2.0
|
30 fmol/μL Gal4DB-mCh |
2.0 |
---
|
30 fmol/μL ATF2 |
2.0 |
---
|
10x T4 Ligation buf (NEB) |
1.0 |
1.0
|
T4 PNK (NEB) |
0.5 |
0.5
|
dH2O |
2.5 |
6.5
|
|
10.0 μL |
10.0 μL
|
- Thermal cycler: program the following...
- Incubate at 37°C/ 30 min.
- Heat-inactivate PNK at 65°C/ 20 min.
LCR Reactions
- MV10 + Gal4DB-mCh + ATF2
- MV10 (neg. ctrl.)
- 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 multiplier = number of LCR reactions
Master Mix (in 1.5 mL tube)
Reagent |
Volume |
MM x3
|
Oligo Bridge 1 |
2.0 |
6.0
|
Oligo Bridge 2 |
2.0 |
6.0
|
Oligo Bridge 3 |
2.0 |
6.0
|
10X Ampligase Buffer |
2.0 |
6.0
|
Ampligase |
1.0 |
1.0
|
dH2O |
3.0 |
9.0
|
|
20.0 μL
|
Add master mix into 10 μL PNK DNA
Reagent |
Volume |
Rxn1 |
Rxn2
|
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.)
|