User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/20

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| 8. MV9(X dp)/ 25 ng ||  
| 8. MV9(X dp)/ 25 ng ||  
 +
|-
 +
| 9. MV9(X no dp)/ 25 ng + T4 ligase ||  
 +
|-
 +
| 10. MV9(X no dp)/ 25 ng ||  
|}
|}
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
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| &nbsp;            || 1    || 2    || 3    || 4    || 5    || 6    || 7    || 8  
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| &nbsp;            || 1    || 2    || 3    || 4    || 5    || 6    || 7    || 8 ||  9  || 10 
|-
|-
-
| Insert DNA        || 0.5  || 1.0  || 2.0  || ---  || 0.5  || 1.0  || 2.0  || ---   
+
| Insert DNA        || 0.5  || 1.0  || 2.0  || ---  || 0.5  || 1.0  || 2.0 || ---  || --- || ---   
|-
|-
-
| Vector DNA        || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0   
+
| Vector DNA        || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || ---
|-
|-
-
| 2x lgn buf (Roche) || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0   
+
| 2x lgn buf (Roche) || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0   
|-
|-
-
| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0   
+
| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0   
|-
|-
-
| dH<sub>2</sub>O    || 2.5  || 2.0  || 1.5 || 3.0  || 2.5  || 2.0  || 1.5 || 3.0   
+
| dH<sub>2</sub>O    || 2.5  || 2.0  || 1.0 || 3.0  || 2.5  || 2.0  || 1.0 || 3.0  || 3.0  || 4.0
|-
|-
-
| &nbsp;            || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL  
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| &nbsp;            || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL  
|}
|}

Revision as of 20:29, 20 March 2013

Karmella's BioBrick Cloning Main project page
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03/20/13

  • Oligo annealing, round 2
  • Assembly: annealed part into MV2



Assemblies

  • New strategy for mammalian vector:
    • Insert K-XbaI-NLS-6his-stop into the puro vector that has a deleted promoter. This way, the vector can be customized to carry different promoters.
    • Insertion of the ds oligo should destroy the vector's XbaI site
    • Will need to check inserts for copy number and orientation


  • Oligo annealing
  • Redo oligo annealing with 99.9 and 97.9°C starting temperatures...
  • Previous Thermal cycling (3/19/13)
    • 99.9, 10 min.
    • [99.9, 1 min., -2°C; 90.0, 1 min., -2°C] x35
    • 25°C, ∞
    • Labeled tubes A1, A2; Stored on bench o/n
  • New Thermal cycling (3/20/13)
    • 99.9, 10 min.
    • [99.9, 1 min., -2°C; 97.9, 1 min., -2°C] x36
    • 25°C, ∞
    • Labeled tubes A3, A4


  • Digest (Fermentas FD)
    • MV2, XbaI only
Reagent Volume
DNA (plasmid) 20.0 μL
10x buffer 3.0
XbaI 2.0
dH2O 5.0
  30 μL --> 37°C/ ~15 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. MV2 (XbaI) 0.081 1.841 81.3


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) 6.2 μL (500 ng)
10x buffer d.p. 2.0
rapid alk. phosphatase 1.0
dH2O 10.8
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) 03/21/13
1. A1 (ds oligo S/S)/size, 0.5 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
2. A1 (ds oligo S/S)/size, 1.0 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
3. A1 (ds oligo S/S)/size, 2.0 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
4. MV9(X dp)/ 25 ng  
5. A3 (ds oligo S/S)/size, 0.5 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
6. A3 (ds oligo S/S)/size, 1.0 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
7. A3 (ds oligo S/S)/size, 2.0 μL + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
8. MV9(X dp)/ 25 ng  
9. MV9(X no dp)/ 25 ng + T4 ligase  
10. MV9(X no dp)/ 25 ng  
  1 2 3 4 5 6 7 8 9 10
Insert DNA 0.5 1.0 2.0 --- 0.5 1.0 2.0 --- --- ---
Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 ---
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 2.5 2.0 1.0 3.0 2.5 2.0 1.0 3.0 3.0 4.0
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL



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