Haynes Lab:Notebook/Engineering PC-TFs/2013/02/08

From OpenWetWare

< Haynes Lab:Notebook | Engineering PC-TFs | 2013 | 02(Difference between revisions)
Jump to: navigation, search
(→Summary)
Current revision (16:13, 9 February 2013) (view source)
(→Summary)
 
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-
==Summary==
+
==2/8/2013==
* * '''Perform BsmBI/ T4 ligase mediated assembly'''
* * '''Perform BsmBI/ T4 ligase mediated assembly'''
* BsmBI cuts the DNA fragments and creates complementary overhangs.
* BsmBI cuts the DNA fragments and creates complementary overhangs.
Line 59: Line 59:
| Total|| 10.0 || 10.0
| Total|| 10.0 || 10.0
|}
|}
 +
 +
 +
<font size=3>'''Bacterial transformation'''</font>
 +
* Added total volume (10.0 μL) to 50 μL chemically competent cells  (e.g., BL21) in a 2.0 mL tube.
 +
* Incubated on ice for 2 min., heat shock at 42°C for exactly 45 sec., and placed on ice.
 +
* Added 800 μL sterile SOC medium.
 +
* Grow with shaking at 37°C for 30 min.(''Taped tubes to the shaker rack.'') 
 +
* Pelleted the cells at top speed in a microcentrifuge for 3 min. at room temperature.
 +
* Discarded the supernatant. Resuspended the cells in 100 μL LB + antibiotic.
 +
* Plate cells on pre-warmed LB agar + antibiotic. (''I plated 100 μL of the cells. Then put beads on plates and shook.) Protocol didn't specify.'')  Grow overnight at 37°C.
 +
* Check results tomorrow.
 +
 +
[[Image:plates_1_8_2013.jpg|400px||left|]]
 +
 +
*''Still nothing. I'm going to start everything over and see if I can troubleshoot. Vi should be working on Type IIS Assembly this week. So I should have something going by the end of next week.''

Current revision

Engineering PC-TFs Main project page
Previous entry      Next entry

2/8/2013

  • * 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 2 3
gg2 pSB1A3 1.0 1.0 1.0
gg3 hPCD 1.0 1.0 1.0
BL02 1.0 --- ---
BL03 --- 1.0 ---
BL04 --- --- 1.0
10x ligase buffer 1.0 1.0 1.0
NEB T4 lgase 0.25 0.25 1.0
BsmBI 0.5 0.5 0.5
dH2O 5.25 5.25 5.25
Total 10.0 10.0


Bacterial transformation

  • Added total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
  • Incubated on ice for 2 min., heat shock at 42°C for exactly 45 sec., and placed on ice.
  • Added 800 μL sterile SOC medium.
  • Grow with shaking at 37°C for 30 min.(Taped tubes to the shaker rack.)
  • Pelleted the cells at top speed in a microcentrifuge for 3 min. at room temperature.
  • Discarded the supernatant. Resuspended the cells in 100 μL LB + antibiotic.
  • Plate cells on pre-warmed LB agar + antibiotic. (I plated 100 μL of the cells. Then put beads on plates and shook.) Protocol didn't specify.) Grow overnight at 37°C.
  • Check results tomorrow.
  • Still nothing. I'm going to start everything over and see if I can troubleshoot. Vi should be working on Type IIS Assembly this week. So I should have something going by the end of next week.



Retrieved from "http://openwetware.org/wiki/Haynes_Lab:Notebook/Engineering_PC-TFs/2013/02/08"
Personal tools