Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/Entry Base"

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(mm/dd/yy)
(mm/dd/yy)
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
# 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)
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> Measure conc.'s
+
* Measure conc.'s
 
{| {{table}} cellspacing="3" <!-- [DNA] table -->
 
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|-
+
|- bgcolor=#cfcfcf  
| bgcolor=#cfcfcf | Sample  
+
| Sample || OD260 || 260/280 || ng/μL
| bgcolor=#cfcfcf | OD260
 
| bgcolor=#cfcfcf | 260/280
 
| bgcolor=#cfcfcf | ng/μL
 
 
|-
 
|-
 
| 1. Digested part (a/b) || --- || --- || ---
 
| 1. Digested part (a/b) || --- || --- || ---
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> Dephosphorylation (Roche)
+
* Dephosphorylation (Roche)
 
{| {{table}} cellspacing="3" <!-- Dephos table -->
 
{| {{table}} cellspacing="3" <!-- Dephos table -->
 
|-
 
|-
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> Ligations
+
* Ligations
 
{| {{table}} cellspacing="3" <!-- Ligations table -->
 
{| {{table}} cellspacing="3" <!-- Ligations table -->
 
|- bgcolor=#cfcfcf
 
|- bgcolor=#cfcfcf

Revision as of 13:35, 1 December 2012

Owwnotebook icon.pngKarmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page

mm/dd/yy

  • Line item 1
  • Line item 2



Minipreps

  • Check with E/P digests
Reagent Volume Expected:
1. BB 1 = size
2. BB2 = size
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

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