Haynes Lab:Notebook/Engineering PC-TFs/2013/02/13

From OpenWetWare

< Haynes Lab:Notebook | Engineering PC-TFs | 2013 | 02(Difference between revisions)
Jump to: navigation, search
(→2/13/13)
Current revision (01:18, 16 February 2013) (view source)
(→Summary)
 
Line 40: Line 40:
| gg2 pSB1A3 || 1.0  
| gg2 pSB1A3 || 1.0  
|-
|-
-
| gg3 hPCD || 1.0  
+
| gg3 hPCD || 1.0
 +
|-
| BL01 || 1.0
| BL01 || 1.0
|-
|-

Current revision

Engineering PC-TFs Main project page
Previous entry      Next entry

Summary

  • 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 (1 μL) 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
Reagent 1
gg2 pSB1A3 1.0
gg3 hPCD 1.0
BL01 1.0
10x ligase buffer 1.0
NEB T4 lgase 0.25
BsmBI 0.5
dH2O 5.25
Total 10.0

Could not perform bacterial transformation because SOC medium was in freezer. Vi performed BsmBI/T4 ligase and bacterial transformation for BL01 and hPCD and there were no colonies on plates.


Retrieved from "http://openwetware.org/wiki/Haynes_Lab:Notebook/Engineering_PC-TFs/2013/02/13"
Personal tools