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

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'''Assemblies'''
'''Assemblies'''
-
# KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(X/P)/4529
+
# KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(S/P)/4529
> Digests (Fermentas FD)<br>
> Digests (Fermentas FD)<br>
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| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
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|-
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| 1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng || <font color="blue">KAH3/MV2 #:1 (Pick #)</font>
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| 1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng || <font color="blue">KAH3/MV2 >10:1 (Pick 2)</font>
|-
|-
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| 2. MV2 (S/P)/ 25 ng || &nbsp;
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| 2. MV2 (S/P)/ 25 ng || zero colonies
|}
|}

Current revision

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/(S/P)/4529

> Digests (Fermentas FD)


Reagent Volume   Hover name
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 >10:1 (Pick 2)
2. MV2 (S/P)/ 25 ng zero colonies
  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
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar



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