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From 2008.igem.org

Contents 1 Protocol 1.1 Hybridization of Parts 1.2 DNA Ligation 2 Results 3 Discussion

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 1 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 -20^oC

Reference: Quick Ligation Kit from NEB.

Results

Discussion

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