User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/12: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(4 intermediate revisions by the same user not shown)
Line 9: Line 9:
=01/12/2013=
=01/12/2013=
Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21
Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21
* The 4 μL of C-His Vector from stock is diluted with 20 μL of nuclease free water, then cut with E/P and purified from the gel.   
* The 4 μL of C-His Vector from stock is diluted with 16 μL of nuclease free water, then cut with E/P and purified from the gel.   
* Ligations
* Ligations
{| {{table}} cellspacing="3" <!-- Ligations table -->
{| {{table}} cellspacing="3" <!-- Ligations table -->
Line 26: Line 26:
|}
|}


* Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2  || 3  ||
| &nbsp;            || 1    || 2  || 3  || 4 || 5
|-
|-
| Insert DNA        || 3.12 || 3.12 || --- ||
| Insert DNA        || 2.34 || 2.34 || 2.05 || 2.05 || ---
|-
|-
| Vector DNA        || 2.94 || 2.94 || 2.94 ||
| Vector DNA        || 1.92 || 1.92 || 1.92 || 1.92 || 1.92
|-
|-
| 2x lgn buf (Roche) || 7.06 || 7.06 || 5.0 ||
| 2x lgn buf (Roche) || 5.26 || 5.26 || 5.0 || 5.0 || 5.0
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0 ||
| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0 || 1.0 || 1.0
|-
|-
| dH<sub>2</sub>O    || 0 || 0 || 0.88 ||
| dH<sub>2</sub>O    || 0 || 0 || 0.03 || 0.03 || 2.08
|-
|-
| &nbsp;            || 14.12 μL || 14.12 μL || 10μl ||
| &nbsp;            || 10.52 μL || 10.52 μL || 10μl || 10μL || 10μL
|}
|}
* The incubation time for the ligation process was 30min at room temperature.
* Fast transformation, 30 min on ice.

Revision as of 14:52, 12 January 2013

PcTF Subcloning in E. coli <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>

01/12/2013

Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21

  • The 4 μL of C-His Vector from stock is diluted with 16 μL of nuclease free water, then cut with E/P and purified from the gel.
  • Ligations
Ligation
1. E. coli BL-21 KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
2. E. coli DH5α-T KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
3. E. coli BL-21 KAH112(E/P)/size, 22.8 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
4. E. coli DH5α-T KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
5. C-His(E/P)/ 26 ng (Control Plate)
  • Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
  1 2 3 4 5
Insert DNA 2.34 2.34 2.05 2.05 ---
Vector DNA 1.92 1.92 1.92 1.92 1.92
2x lgn buf (Roche) 5.26 5.26 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 0 0 0.03 0.03 2.08
  10.52 μL 10.52 μL 10μl 10μL 10μL
  • The incubation time for the ligation process was 30min at room temperature.
  • Fast transformation, 30 min on ice.
Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/01/12&oldid=667075"