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

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'''Assemblies'''
'''Assemblies'''
* 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 (MV2, puro). This way, the vector can be customized to carry different promoters.
** Insertion of the ds oligo should destroy the vector's XbaI site
** Insertion of the ds oligo should destroy the vector's XbaI site
** Will need to check inserts for copy number and orientation
** Will need to check inserts for copy number and orientation
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* '''Oligo annealing'''
* '''Oligo annealing'''
* See [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/19 03/19/13] for details about the oligos and reaction set-up
* Redo oligo annealing with 99.9 and 97.9°C starting temperatures...
* Redo oligo annealing with 99.9 and 97.9°C starting temperatures...


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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
| rowspan="7" | [[Image:KAH032013_gel1.jpg‎|100px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA (plasmid) || 20.0 μL
| DNA (plasmid) || 20.0 μL
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|}
|}


* Gel purify using the Zymo DNA Gel Recovery kit.
* Elute with 20 μL dH<sub>2</sub>O


* Measure conc.'s
 
 
* '''Measure conc.'''
{| {{table}} cellspacing="3" <!-- [DNA] table -->
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf  
|- bgcolor=#cfcfcf  
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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
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* Ligations
* '''Ligations'''
{| {{table}} cellspacing="3" <!-- Ligations table -->
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- bgcolor=#cfcfcf
|- bgcolor=#cfcfcf
| 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)/size, 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="red">MV9 failed</font>
|-
|-
| 2. A1 (ds oligo S/S)/size, ''1.0 μL'' + MV2(X dp)/size, 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="red">MV9 failed</font>
|-
|-
| 3. A1 (ds oligo S/S)/size, ''2.0 μL'' + MV2(X dp)/size, 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="red">MV9 failed</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)/size, 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="red">MV9 failed</font>
|-
|-
| 6. A3 (ds oligo S/S)/size, ''1.0 μL'' + MV2(X dp)/size, 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="red">MV9 failed</font>
|-
|-
| 7. A3 (ds oligo S/S)/size, ''2.0 μL'' + MV2(X dp)/size, 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="red">MV9 failed</font>
|-
|-
| 8. MV9(X dp)/ 25 ng || &nbsp;
| 8. MV9(X dp)/ 25 ng || &nbsp;
|-
| 9. MV9(X no dp)/ 25 ng + T4 ligase || <font color="blue">Re-ligation successful, ~40 colonies</font>
|-
| 10. MV9(X no dp)/ 25 ng || &nbsp;
|}
|}


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    || 3    || 4    || 5    || 6    || 7    || 8  ||  9  || 10 
|-
|-
| Insert DNA        || 0.5  || ---  ||
| Insert DNA        || 0.5 || 1.0  || 2.0 || ---  || 0.5  || 1.0  || 2.0  || ---  || ---  || --- 
|-
|-
| Vector DNA        || 1.0  || 1.0  ||
| Vector DNA        || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 0.3  || 0.3
|-
|-
| 2x lgn buf (Roche) || 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  ||
| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || --- 
|-
|-
| dH<sub>2</sub>O    || ### || ### ||
| dH<sub>2</sub>O    || 2.5 || 2.0 || 1.0  || 3.0  || 2.5  || 2.0  || 1.0  || 3.0  || 3.7  || 4.7
|-
|-
| &nbsp;            || 10 μL || 10 μL ||
| &nbsp;            || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL
|}
|}


* 10 min./ room temp
* Add 30 μL DH5α; 5 min/ ice
* Plate on 100 μg/mL amp; incubate at 37°C
----
3/21/13<br>
Results for plates 9 and 10
[[Image:KAH032113_plates1.jpg‎|300px|Hover name]]




Revision as of 14:50, 21 March 2013

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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 (MV2, puro). 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
  • See 03/19/13 for details about the oligos and reaction set-up
  • 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 Hover name
30 μL/lane, 1% agarose; Ladder
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 failed
2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 failed
3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng MV9 failed
4. MV9(X dp)/ 25 ng  
5. A3 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng MV9 failed
6. A3 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 failed
7. A3 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng MV9 failed
8. MV9(X dp)/ 25 ng  
9. MV9(X no dp)/ 25 ng + T4 ligase Re-ligation successful, ~40 colonies
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 0.3 0.3
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 ---
dH2O 2.5 2.0 1.0 3.0 2.5 2.0 1.0 3.0 3.7 4.7
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL
  • 10 min./ room temp
  • Add 30 μL DH5α; 5 min/ ice
  • Plate on 100 μg/mL amp; incubate at 37°C

3/21/13

Results for plates 9 and 10

Hover name



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