Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10"

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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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'''Golden gate trial 1'''
 
'''Golden gate trial 1'''
 
* Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
 
* Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
* Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and seed 2 mL liquid cultures.
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* Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.
  
  
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PCR
 
PCR
 
* 95°C, 3 min.
 
* 95°C, 3 min.
* [95°C, 3 min.; 95°C, 3 min.; 95°C, 3 min.] x35
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* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
 
* 72°C, 3 min.
 
* 72°C, 3 min.
 
* 4°C, ∞
 
* 4°C, ∞
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Dilutions
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Dilutions: to final concentration of 20 fmol/μL
 
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
 
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
 
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* Golden Gate Reactions
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Golden Gate Reactions
 
# hPCD + BL01 + pSB1A3
 
# hPCD + BL01 + pSB1A3
 
# pSB1A3
 
# pSB1A3
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|}
 
|}
  
Thermal cycler
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Thermal cycling
 
* [45°C, 2 min.; 16°C, 5 min.] x25
 
* [45°C, 2 min.; 16°C, 5 min.] x25
 
* 60°C, 20 min.
 
* 60°C, 20 min.
 
* 80°C, 20 min.
 
* 80°C, 20 min.
 +
* 4°C, ∞
  
  

Latest revision as of 21:20, 26 September 2017

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

01/10/13

  • Violacein: successful growth; re-streak
  • Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
  • Golden gate trial 2: new PCR primers



Violacein

  • Plate 1: Kan plasmid
  • Plate 2: Amp plasmid (violacein exp.)



Golden gate trial 1

  • Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
  • Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.



Golden gate trial 2

  • Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region
Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520
Hover name
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 22.8
  50.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞


Purification

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. pSB1A3 0.126 1.888 77.357
2. hPCD 0.069 2.192 17.025
3. BL01 0.121 1.898 66.878


Dilutions: to final concentration of 20 fmol/μL

DNA length (bp) ng/μL Vol. + dH2O
pSB1A3 2000 77.4 6.7 13.3
hPCD 186 17.0 2.8 17.2
BL01 2520 66.9 9.8 10.2


Golden Gate Reactions

  1. hPCD + BL01 + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycling

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞