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

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(11/18/09)
(11/18/09)
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--> Specific notes<br>
--> Specific notes<br>
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn table -->
+
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
 +
|-valign="top"
 +
| <u>Reagent</u> || <u>Volume</u>
 +
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
 +
| rowspan="7" | <!-- Gel image [[Image:KAH_111909_gel1.jpg|400px|E/P Digest1 11/19/09]] -->
|-
|-
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| <u>Reagent</u> || <u>Volume</u>
+
| DNA(plasmid) || 2.0 μL
|-
|-
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| DNA (plasmid) || up to 25 μL
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| 10X buffer || 1.5
|-
|-
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| 10x buffer || 3.0
+
| EcoRI || 1.0
|-
|-
-
| enzyme 1 || 1.0
+
| PstI || 1.0
|-
|-
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| enzyme 2 || 1.0
+
| dH<sub>2</sub>O || 9.5
|-
|-
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| dH<sub>2</sub>O || ---
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| &nbsp; || 15 μL --> 37°C/ ~15 min.
-
|-
+
-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
+
|}
|}

Revision as of 10:37, 20 November 2009

Project name Main project page

11/18/09

  • Unfinished line item
  • ✓ Finished line item

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 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.


> 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 x:y (Pick 2#)
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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