ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry
LCR Assembly
PNK treatment
Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups
Procedure:
- 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)
- OPTIONAL - Digest the template DNA: Include 1 μL FastDigest DpnI in the PNK reaction. DpnI will cut only methylated DNA that came from a bacterial cell (plasmid template) and not the synthetic PCR product.
PNK reaction
| Reagent |
Volume
|
| Clean PCR or dsDNA |
up to 8.5 μL
|
| 10x T4 Ligation buf (NEB) |
1.0
|
| T4 PNK (NEB) |
0.5
|
| dH2O |
x μL
|
| |
10.0 μL
|
- Incubate at 37°C/ 30 min.
- Heat-inactivate PNK at 65°C/ 20 min.
|
Open Chromatin
