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

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(Autocreate 2013/03/20 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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----
----
'''Minipreps'''<br>
'''Assemblies'''
* Check with E/P digests
# MV9: 5' part/(a/b)/size + 3' part/(c/d)/size
 
 
'''Oligo annealing'''
# New BB 1


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
|- valign="top"
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
| bgcolor=#cfcfcf | Reagent
| 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 annealing buffer || 2.0
|-
|-
| 10X buffer || 1.5
| dH<sub>2</sub>O || ---
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|}
|}


----
'''Assemblies'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size




* Digests (Fermentas FD)
* Digests (Fermentas FD)
** Specific notes
** MV2, XbaI only


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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| 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:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
|-
|-
| DNA (plasmid) || up to 25 μL
| DNA (plasmid) || 20.0 μL
|-
|-
| 10x buffer || 3.0
| 10x buffer || 3.0
|-
|-
| enzyme 1 || 1.0
| XbaI || 2.0
|-
| enzyme 2 || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 5.0
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 30 μL --> 37°C/ ~30 min.
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| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. MV2 (XbaI) || 0.081 || 1.841 || 81.3
|-
| 2. Digested part (c/d) || --- || --- || ---
|}
|}


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| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| DNA (clean digest) || 6.2 μL (500 ng)
|-
|-
| 10x buffer d.p. || 2.0
| 10x buffer d.p. || 2.0
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| phosphatase || 1.0
| phosphatase || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 10.8
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL

Revision as of 13:26, 20 March 2013

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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mm/dd/yy

  • Line item 1
  • Line item 2


Assemblies

  1. MV9: 5' part/(a/b)/size + 3' part/(c/d)/size


Oligo annealing

  1. New BB 1
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


  • Digests (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/ ~30 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
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) 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



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