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

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(03/20/13)
Line 17: Line 17:
* New strategy for mammalian vector:  
* 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.
** 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
Line 89: Line 91:
| 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. insert(Oligo S/S)/size, ## ng + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
+
| 1. A1 (ds oligo S/S)/size, ''0.5 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
|-
|-
-
| 2. insert(Oligo S/S)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
+
| 2. A1 (ds oligo S/S)/size, ''1.0 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
|-
|-
-
| 3. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
+
| 3. A1 (ds oligo S/S)/size, ''2.0 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
|-
|-
-
| 4. vector(c/d)/ ## ng || &nbsp;
+
| 4. MV9(X dp)/ 25 ng || &nbsp;
 +
|-
 +
| 5. A3 (ds oligo S/S)/size, ''0.5 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
 +
|-
 +
| 6. A3 (ds oligo S/S)/size, ''1.0 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
 +
|-
 +
| 7. A3 (ds oligo S/S)/size, ''2.0 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
 +
|-
 +
| 8. MV9(X dp)/ 25 ng || &nbsp;
|}
|}
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| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
-
| Insert DNA        || ### || ---  ||
+
| Insert DNA        || 0.5 || ---  ||
|-
|-
-
| Vector DNA        || ### || ### ||
+
| Vector DNA        || 1.0 || 1.0 ||
|-
|-
-
| 2x lgn buf (Roche) || ### || ### ||
+
| 2x lgn buf (Roche) || 5.0 || 5.0 ||
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0  ||
Line 111: Line 121:
| dH<sub>2</sub>O    || ###  || ###  ||
| dH<sub>2</sub>O    || ###  || ###  ||
|-
|-
-
| &nbsp;            || # μL || # μL ||
+
| &nbsp;            || 10 μL || 10 μL ||
|}
|}
-
----
 
-
'''Oligo annealing'''
 
-
# New BB 1
 
-
# New BB 2
 
-
 
-
{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
 
-
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
 
-
|-
 
-
| 10x annealing buffer || 2.0
 
-
|-
 
-
| dH<sub>2</sub>O || ---
 
-
|-
 
-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
 
-
|}
 

Revision as of 16:49, 20 March 2013

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

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  
  1 2
Insert DNA 0.5 ---
Vector DNA 1.0 1.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  10 μL 10 μL



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