Knight:Annealing and cloning oligos

in progress, this protocol hasn't worked yet; use at your own risk!!!

Overview

This is a protocol to anneal and ligate oligo's to construct short DNA fragments for cloning (~100 bp)

Materials

• 3 oligo pairs with 4-5 base pair overlaps
  • Austin suggests 6 bp overlaps.
• T4 DNA ligase
• T4 DNA ligase buffer
• T4 polynucleotide kinase

Procedure

1. Resuspend primers.
2. Kinase treat each oligo using the following reaction mix for each primer.
   • 2 μL primer (5 μM final concentration)
   • 1 μL 10X T4 DNA ligase buffer
   • 6.5 μL H₂O
   • 0.5 μL T4 polynucleotide kinase
3. Incubate at 37 °C for 1 hr 30 mins
4. Heat kill at 65 °C for 20 mins
5. Mix all 10μL of kinase treated oligo with its pair to make duplex oligos.
6. Heat to 70-75 °C and cool to anneal oligo pairs.
7. Mix all 20 μL of each duplex together in one tube.
8. Make up ligation reaction
   • 1 μL of the oligo mix
   • 50 ng digested vector backbone
     • The assembled oligo construct should have complementary ends to the prepared vector
   • 1 μL 10X T4 DNA ligase buffer
   • H₂O to 10 uL
9. Add 0.5 μL T4 DNA ligase
10. Incubate for 1hr 30 mins at 16°C.
11. Transform.

Notes

• I did not include 5' phosphates on the flanking primers to decrease the likelihood of forming multimers.
• Pete Carr recommends PCR construction and assembly since ligation methods can be unreliable.
• Too much ligase may adversely affect the reaction.