User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/19

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(03/19/13)
Current revision (17:17, 22 March 2013) (view source)
(03/19/13)
 
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==03/19/13==
==03/19/13==
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* Oligo annealing for new mammalian vector based on MV9
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* Oligo annealing for new mammalian vector
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# KAH3/MV2(X) + K-XbaI-NLS-his-stop
# KAH3/MV2(X) + K-XbaI-NLS-his-stop
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* K-XbaI-NLS-his-st 1
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* K-XbaI-NLS-6his-st 1
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* K-XbaI-NLS-his-st 2
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* K-XbaI-NLS-6his-st 2
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* K-XbaI-NLS-his-st 3
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* K-XbaI-NLS-6his-st 3
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* K-XbaI-NLS-his-st 4
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* K-XbaI-NLS-6his-st 4
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{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
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{| {{table}} <!-- Oligo annealing rxn table -->
| DNA (oligos, 100 μM) || 4x 3 μL ea. = 12.0
| DNA (oligos, 100 μM) || 4x 3 μL ea. = 12.0
|-
|-
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* 25°C, ∞
* 25°C, ∞
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'''Vector Digest'''
 
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Current revision

Karmella's BioBrick Cloning Main project page
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03/19/13

  • Oligo annealing for new mammalian vector



Oligo annealing

  • Note: made fresh 10x annealing buffer
  1. KAH3/MV2(X) + K-XbaI-NLS-his-stop
  • K-XbaI-NLS-6his-st 1
  • K-XbaI-NLS-6his-st 2
  • K-XbaI-NLS-6his-st 3
  • K-XbaI-NLS-6his-st 4
DNA (oligos, 100 μM) 4x 3 μL ea. = 12.0
10x annealing buffer 2.0
dH2O 6.0
  20 μL

Thermal Cycling (try new thermal cycling approach)
Multigene Optimax Thermal Cycler

  • 99.9 °C, 10 min.
  • [99.9°C, temp. increment -2.0°C, 1 min.; 90.0°C, temp. increment -2.0°C, 1 min.] x35
  • 25°C, ∞



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