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.

Materials

• 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. See notes for more information.

Oligo 1:    5' ---RE site-------------------------------- 3'
Oligo 2:                                        3' --------------------------------RE site--- 5'

• Klenow 3'[math]\displaystyle{ \rightarrow }[/math]5' exo^- polymerase
• dNTPs (25 mM each dNTP in stock)
• Restriction enzyme(s)
• Restriction enzyme buffer
• BSA

Calculating amount of oligo for reaction

This should be checked for errors -Reshma 19:03, 12 May 2005 (EDT)

[math]\displaystyle{ \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock} }[/math]

Procedure

1. Dilute the two oligos to a concentration of 10 or 25 μM using H₂O
2. 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
3. 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.
4. Add 1 μL Klenow 3'[math]\displaystyle{ \rightarrow }[/math]5' exo^- polymerase to mixture.
   Vortex polymerase before pipetting to ensure it is well-mixed.
5. Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP).
   Recommend using a thermal cycler for the following incubation steps.
6. Incubate 1 hr at 37°C.
7. Heat inactivate polymerase by incubating at 75°C for 20 minutes.
   See Restriction Digest for more information on the following steps.
8. Add 1 μL restriction enzyme(s) to mixture.
9. Incubate for a minimum of 2 hrs.
10. Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.
11. Purify DNA as necessary

Notes

Once oligos become about around 50-60 bp in length, there can often be problems with errors or deletions in the primers. Therefore, it might be worth ordering your primers with an extra purification step such as PAGE. Invitrogen custom primers offers this service for an extra fee.

References

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