Team:Hawaii/Initial Synth. Oligo Assembly

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(Difference between revisions)
(New page: ==Protocol== ===Hybridization of Parts=== #Mix: #:3 μl 100 µM sense oligo #:3 μl 100 µM anti-sense oligo #:3 μl 10 x PNK (polynucleotide kinase) buffer #:2 μl 10mM ATP #:2 μl T...)
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#Incubate at room temperature for 5 minutes.  
#Incubate at room temperature for 5 minutes.  
#Cool on ice then transform, or store at -20<sup>o</sup>C
#Cool on ice then transform, or store at -20<sup>o</sup>C
 +
 +
:::''Reference: Quick Ligation Kit from NEB.''

Revision as of 03:52, 4 July 2008

Contents

Protocol

Hybridization of Parts

  1. Mix:
    3 μl 100 µM sense oligo
    3 μl 100 µM anti-sense oligo
    3 μl 10 x PNK (polynucleotide kinase) buffer
    2 μl 10mM ATP
    2 μl T4 polynucleotide kinase (PNK)
    17 μl distilled water
    Total volume = 30 μl
  2. Incubate at 37C for 1.5 hours.
  3. Add 4 μl 0.5 M NaCl.
  4. Place in boiling water bath for 2 min., then remove water bath from the heat source and allow the reaction (still in the water bath) to cool to room temperature (approx. 30 minutes)
Reference: Pam Silver Lab. [http://openwetware.org/wiki/Silver:_Oligonucleotide_Inserts| Oligonucleotide Inserts.]

DNA Ligation

  1. Combine 4 ul of construct one with 4 ul of construct 2.
  2. Add 2 ul of nanopure H20.
  3. Add 10 ul of 2x quick ligation reaction Buffer. In order to avoid shearing the DNA, mix by resuspending very slowly.
  4. Add 1 ul of Quick T4 DNA ligase and mix thoroughly by resuspending very slowly.
  5. Incubate at room temperature for 5 minutes.
  6. Cool on ice then transform, or store at -20oC
Reference: Quick Ligation Kit from NEB.


Results

Discussion

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