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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
 
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
 
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Latest revision as of 21:33, 26 September 2017

Owwnotebook icon.pngKarmella's BioBrick Cloning Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

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