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.

Also see Knight:Annealing and primer extension with Taq polymerase for generation for short DNA fragments for TA cloning.

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 notes for information on cutting near the ends of linear DNA fragments.  See  notes for more information on primer ordering.

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


 * Klenow 3'$\rightarrow$5' exo- polymerase
 * dNTPs (25 mM each dNTP in stock)
 * Restriction enzyme(s)
 * Restriction enzyme buffer
 * BSA

Calculating amount of oligo for reaction
$$ \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock}$$

Procedure

 * 1) Dilute the two oligos to a concentration of 10 or 25 &mu;M using H2O
 * 2) Mix the following in a 0.6 mL sterile tube
 * 3) *10 &mu;L 10X restriction enzyme buffer
 * 4) *1 &mu;L 100X BSA
 * 5) *X &mu;L oligo 1 (typically 1 &mu;g or more)
 * 6) *Y &mu;L oligo 2 (typically 1 &mu;g or more)
 * 7) *(87 - X - Y) &mu;L deionized sterile H2O
 * 8) Anneal the two oligos together by either placing the mixture in a thermal cycler (MJ Research, PTC-200) at 94&deg;C for 5 mins, a cool down for 0.1&deg;C/sec to 5&deg;C below the melting temperature of the primers, hold that temperature for 5 mins, then cool down at 0.1&deg;C/sec to 37&deg;C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.
 * 9) Add 1 &mu;L Klenow 3'$$\rightarrow$$5' exo- polymerase to mixture. Vortex polymerase before pipetting to ensure it is well-mixed.
 * 10) Add 1 &mu;L dNTPS (equal to 0.25 mM final concentration of each dNTP). Recommend using a thermal cycler for the following incubation steps.
 * 11) Incubate 1 hr at 37&deg;C.
 * 12) Heat inactivate polymerase by incubating at 75&deg;C for 20 minutes. This inactivation temperature might be higher than the melting temperature of your annealed and extended primers.  It may be prudent to ramp the temperature down from 75&deg;C. See Restriction Digest for more information on the following steps.
 * 13) Add 1 &mu;L restriction enzyme(s) to mixture.
 * 14) Incubate for a minimum of 2 hrs.
 * 15) Heat inactivate restriction enzyme by incubating at 80&deg;C for 20 mins.
 * 16) Purify DNA as necessary