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

From OpenWetWare
Jump to navigationJump to search
Line 21: Line 21:




'''PNK treatment'''<br>
PNK treatment<br>
Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups
Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups
* Dilute the purified dsDNA to 30 fmol/μL (30 nM)
* Dilute the purified dsDNA to 30 fmol/μL (30 nM)
Line 29: Line 29:
* Mix the appropriate amount of dsDNA fragments together and treat with Polynucleotide kinase to add 5'-phosphates (see table below)
* Mix the appropriate amount of dsDNA fragments together and treat with Polynucleotide kinase to add 5'-phosphates (see table below)


PNK reaction
{| {{table}}
{| {{table}}
|-
|-
Line 53: Line 51:
* Heat-inactivate PNK at 65°C/ 20 min.
* Heat-inactivate PNK at 65°C/ 20 min.


 
LCR Reaction




Revision as of 22:23, 3 August 2015

Open Chromatin <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry

  • LCR Assembly


LCR Assembly

Prep the Bridge Oligos
Note: Final conc. in LCR rxn. is 30 nM each

  1. 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
  2. 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



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Daniel_A._Vargas/Notebook/General_lab_notebook/2015/08/04&oldid=895751"