McClean: Anneal and Extend
This protocol describes a simple procedure to annealing and extending two oligos (with homology) to get double-stranded DNA.
- 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)
- Design oligos so that they have ~20bp of overlap.
- Dilute oligos to 25μM from the -80°C stock. Dilute into H2O.
- 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
- 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
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.
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.