User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/20: Difference between revisions

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|   || 30 μL --> 37°C/ ~15 min.
|   || 30 μL --> 37°C/ ~15 min.
|}
|}
* Gel purify using the Zymo DNA Gel Recovery kit.
* Elute with 20 μL dH<sub>2</sub>O




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| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || 6.2 μL (500 ng)
| MV2 (XbaI) || 6.2 μL (500 ng)
|-
|-
| 10x buffer d.p. || 2.0
| 10x buffer d.p. || 2.0
Line 91: Line 95:
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/21/13</font>
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/21/13</font>
|-
|-
| 1. A1 (ds oligo S/S)/size, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
| 1. A1 (ds oligo S/S)/76, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
|-
|-
| 2. A1 (ds oligo S/S)/size, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
| 2. A1 (ds oligo S/S)/76, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
|-
|-
| 3. A1 (ds oligo S/S)/size, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
| 3. A1 (ds oligo S/S)/76, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
|-
|-
| 4. MV9(X dp)/ 25 ng || &nbsp;
| 4. MV9(X dp)/ 25 ng || &nbsp;
|-
|-
| 5. A3 (ds oligo S/S)/size, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
| 5. A3 (ds oligo S/S)/76, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
|-
|-
| 6. A3 (ds oligo S/S)/size, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
| 6. A3 (ds oligo S/S)/76, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
|-
|-
| 7. A3 (ds oligo S/S)/size, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
| 7. A3 (ds oligo S/S)/76, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
|-
|-
| 8. MV9(X dp)/ 25 ng || &nbsp;
| 8. MV9(X dp)/ 25 ng || &nbsp;

Revision as of 19:21, 20 March 2013

Karmella's BioBrick Cloning <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>

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.
  • Gel purify using the Zymo DNA Gel Recovery kit.
  • Elute with 20 μL dH2O


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


  • Dephosphorylation (Roche)
Reagent Volume
MV2 (XbaI) 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)/76, 0.5 μL + MV2(X dp)/4529, 25 ng MV9 #:1 (Pick #)
2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 #:1 (Pick #)
3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng MV9 #:1 (Pick #)
4. MV9(X dp)/ 25 ng  
5. A3 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng MV9 #:1 (Pick #)
6. A3 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 #:1 (Pick #)
7. A3 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 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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