User:Karmella Haynes/Notebook/BioBrick cloning/Entry Base

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(mm/dd/yy)
Current revision (16:35, 1 December 2012) (view source)
(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
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> Digests (Fermentas FD)<br>
 
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--> Specific notes<br>
 
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
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* Digests (Fermentas FD)
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|-valign="top"
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** Specific notes
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| <u>Reagent</u> || <u>Volume</u> || &nbsp;
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| rowspan="9" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
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{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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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] -->
|-
|-
| DNA (plasmid) || up to 25 μL
| DNA (plasmid) || up to 25 μL
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> Measure conc.'s
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* Measure conc.'s
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{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
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{| {{table}} cellspacing="3" <!-- [DNA] table -->
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|-
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|- bgcolor=#cfcfcf
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| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
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| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. Digested part (a/b) || --- || --- || ---
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> Dephosphorylation (Roche)
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* Dephosphorylation (Roche)
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{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
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{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
|-
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| <u>Reagent</u> || <u>Volume</u>
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| DNA (clean digest) || up to 17 μL (500 ng)
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> Ligations
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* Ligations
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{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
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{| {{table}} cellspacing="3" <!-- Ligations table -->
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| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
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|- bgcolor=#cfcfcf
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| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
|-
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
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|}
|}
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{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-

Current revision

Karmella's BioBrick Cloning 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



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