Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/15"

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(Autocreate 2013/01/15 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
(mm/dd/yy)
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==mm/dd/yy==
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==01/15/13==
 
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* Golden gate assembly: test different ligase buffers
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----
 
----
'''Minipreps'''<br>
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'''Golden Gate Assembly optimization'''<br>
* Check with E/P digests
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* Test different buffers
  
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
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|- valign="top" bgcolor=#cfcfcf  
| bgcolor=#cfcfcf | Reagent
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| Buffer || Tris-HCl || MgCl<sub>2</sub> || DTT || ATP
| bgcolor=#cfcfcf | Volume
 
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
 
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
 
|-
 
| DNA(plasmid) || 2.0 μL
 
|-
 
| 10X buffer || 1.5
 
|-
 
| EcoRI || 1.0
 
 
|-
 
|-
| PstI || 1.0
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| 10x Promega || 300 mM (pH 7.8) || 100 mM || 100 mM || 10 mM
 
|-
 
|-
| dH<sub>2</sub>O || 9.5
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| 10x NEB || 500 mM (pH 7.5) || 100 mM || 100 mM || 10 mM
 
|-
 
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
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| 2x Roche  || ? || ? || ? || ? || ?
 
|}
 
|}
  

Revision as of 15:46, 15 January 2013

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01/15/13

  • Golden gate assembly: test different ligase buffers



Golden Gate Assembly optimization

  • Test different buffers
Buffer Tris-HCl MgCl2 DTT ATP
10x Promega 300 mM (pH 7.8) 100 mM 100 mM 10 mM
10x NEB 500 mM (pH 7.5) 100 mM 100 mM 10 mM
2x Roche  ?  ?  ?  ?  ?

Assemblies

  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


  • Digests (Fermentas FD)
    • Specific notes
Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight