Haynes Lab:Notebook/Engineering PC-TFs/2013/01/29

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(→Summary)
m (→Summary)
Line 19: Line 19:
* T4 ligase seals gaps in the phosphodiester DNA backbone.
* T4 ligase seals gaps in the phosphodiester DNA backbone.
{| {{table}}
{| {{table}}
-
|
 
| bgcolor="grey" | Reagent
| bgcolor="grey" | Reagent
| bgcolor="grey" | Vol.
| bgcolor="grey" | Vol.

Revision as of 03:04, 1 February 2013

Engineering PC-TFs Main project page
Previous entry      Next entry

Summary


Digestion/ ligation reaction


  • Dilute the purified PCR product to 20 fmol/μL
  • Measure ng/μL of the purified sample.
  • The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * length in bp * 650 fg/fmol ÷ 1,000,000 fg/ng ÷ measured ng/μL
    • Formula: x = length in bp ÷ measured ng/μL * 0.26
  • Perform BsmBI/ T4 ligase mediated assembly
  • BsmBI cuts the DNA fragments and creates complementary overhangs.
  • Complementary sticky ends anneal via base pairing.
  • T4 ligase seals gaps in the phosphodiester DNA backbone.
Reagent Vol. Thermal cycling
  • [45°C, 2 min.; 16°C 5 min.] x25
  • 60°C, 10 min.
  • 80°C, 20 min.
  • 4°C, ∞
20 fmol of each DNA part up to 8.0
10x T4 ligase buffer (Promega) 1.0
T4 ligase (NEB) 0.25
BsmBI 0.5
dH2O 0.25
  10.0 μL


|}


Retrieved from "http://openwetware.org/wiki/Haynes_Lab:Notebook/Engineering_PC-TFs/2013/01/29"
Personal tools