Knight:Annealing and primer extension with Klenow polymerase
This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp). The DNA fragment is prepared for cloning by restriction digest.
- Two oligos which overlap by ~20 bp and have restriction enzyme sites at the 5' ends as in the diagram below. See Restriction Digest for notes on cutting near the ends of linear DNA fragments.
Oligo 1: 5' ---RE site-------------------------------- 3'
Oligo 2: 3' --------------------------------RE site--- 5'
- Klenow 3'5' exo- polymerase
- dNTPs (25 mM each dNTP in stock)
- Restriction enzyme(s)
- Restriction enzyme buffer
Calculating amount of oligo for reaction
This should be checked for errors -Reshma 19:03, 12 May 2005 (EDT)
- Dilute the two oligos to a concentration of 10 or 25 μM using H2O
- Mix the following in a 0.6 mL sterile tube
- 10 μL 10X restriction enzyme buffer
- 1 μL 100X BSA
- X μL oligo 1 (typically 1 μg or more)
- Y μL oligo 2 (typically 1 μg or more)
- (87 - X - Y) μL deionized sterile H2O
- Anneal the two oligos together by either placing the mixture in a thermal cycler (MJ Research, PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 65°C, 65°C for 5 mins, then a cool down for 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.
- Add 1 μL Klenow 3'
Vortex polymerase before pipetting to ensure it is well-mixed.
5' exo- polymerase to mixture.
- Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP).
Recommend using a thermal cycler for the following incubation steps.
- Incubate 1 hr at 37°C.
- Heat inactivate polymerase by incubating at 75°C for 20 minutes.
See Restriction Digest for more information on the following steps.
- Add 1 μL restriction enzyme(s) to mixture.
- Incubate for a minimum of 2 hrs.
- Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.
- Purify DNA as necessary
W. P. Stemmer, A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164(1):49–53, 1995. PubMed