McClean: Anneal and Extend

Overview

This protocol describes a simple procedure to annealing and extending two oligos (with homology) to get double-stranded DNA.

Materials

• Oligos (25μM; take the standard McClean Lab -80°C stock which is at 100μM in TE and dilute it 1:4 in H₂O)
• Takara PCR reagents (see: Takara PrimeStar PCR)

Protocol

1. Design oligos so that they have ~20bp of overlap.
2. Dilute oligos to 25μM from the -80°C stock. Dilute into H₂O.
3. For one reaction the PCR mix should be:
   • 10μL 5X Takara Buffer
   • 4μL 2.5mM DNTPs
   • 0.5μL Takara polymerase
   • 1 μL 25μM forward oligo
   • 1 μL 25μM reverse oligo
   • 33.5μL H₂O
4. Run in a thermocycler as follows (notice that we do not use many cycles):
   • 94°C for 5 min
   • Repeat the following three lines 2-3 times:
     • 98°C for 10s
     • 53°C for 20s
     • 72°C for 30s
   • 72°C 5 min

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

• Megan N McClean It would probably be better to dilute oligos ordered for this purpose into water and not TE pH8 for the -80°C stock. However, I usually forget to do this and diluting from the TE stock 1:4 for 25μM and running the above protocol usually works really well.

Contact

• Megan N McClean 14:01, 19 September 2012(EDT)

or instead, discuss this protocol.