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

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'''PNK treatment'''
'''PNK treatment'''
* Mix double-stranded DNA
* Mix double-stranded DNA fragments, treat with PNK to add phosphate groups
* 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)
** 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
{| {{table}}
{| {{table}}
|-
|-
|
| Reagent || Volume
|-
|-
|
| Clean PCR or dsDNA || up to 8.5 μL
|-
| 10x T4 Ligation buf (NEB) || 1.0
|-
| T4 PNK (NEB) || 0.5
|-
| dH<sub>2</sub>O || x μL
|-
| &nbsp; || 10.0 μL
|}
|}
* Incubate at 37°C/ 30 min.
* Heat-inactivate PNK at 65°C/ 20 min.




Revision as of 22:03, 3 August 2015

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ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry

  • LCR Assembly


LCR Assembly


PNK treatment

  • 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)
    • 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.




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