User:Karmella Haynes/Notebook/BioBrick cloning/2012/11/30

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(11/30/12)
Line 42: Line 42:
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
|-
|-
-
| 1. KAH3 (X/P) || --- || --- || ---
+
| 1. KAH3 (X/P) || 0.028 || 1.96 || 28.41
|-
|-
-
| 2. MV2 (S/P) || --- || --- || ---
+
| 2. MV2 (S/P) || 0.085 || 1.93 || 85.02
|}
|}
Line 60: Line 60:
| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
-
| Insert DNA        || ### || ---  ||
+
| Insert DNA        || 0.3 || ---  ||
|-
|-
-
| Vector DNA        || ### || ### ||
+
| Vector DNA        || 0.3 || 0.3 ||
|-
|-
| 2x lgn buf (Roche) || 5.0  || 5.0  ||
| 2x lgn buf (Roche) || 5.0  || 5.0  ||
Line 68: Line 68:
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
|-
-
| dH<sub>2</sub>O    || ### || ### ||
+
| dH<sub>2</sub>O    || 3.4 || 3.7 ||
|-
|-
| &nbsp;            || 10 μL || 10 μL ||
| &nbsp;            || 10 μL || 10 μL ||

Revision as of 21:51, 30 November 2012

Karmella's BioBrick Cloning Main project page
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11/30/12

  • Assembly: CMVTetO2:K (KAH3)/ MV; expression vector for Vi's mammalian cell expression experiment



Assemblies

  1. KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(X/P)/4529

> Digests (Fermentas FD)


Reagent Volume   Image:GelImage.jpg
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~15 min.


> Measure conc.'s of gel purified fragments

Sample OD260 260/280 ng/μL
1. KAH3 (X/P) 0.028 1.96 28.41
2. MV2 (S/P) 0.085 1.93 85.02


> Ligations

Ligation Plate results (lig : neg crtl) 11/31/12
1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng KAH3/MV2 #:1 (Pick #)
2. MV2 (S/P)/ 25 ng  
  1 2
Insert DNA 0.3 ---
Vector DNA 0.3 0.3
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 3.4 3.7
  10 μL 10 μL



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