Knight:Annealing and cloning oligos

in progress, this protocol hasn't worked yet; use at your own risk!!!

Overview
This is a protocol to anneal and ligate oligo's to construct short DNA fragments for cloning (~100 bp)

Materials

 * 3 oligo pairs with 4-5 base pair overlaps
 * Austin suggests 6 bp overlaps.
 * T4 DNA ligase
 * T4 DNA ligase buffer
 * T4 polynucleotide kinase

Procedure

 * 1) Resuspend primers.
 * 2) Kinase treat each oligo using the following reaction mix for each primer.
 * 3) *2 &mu;L primer (5 &mu;M final concentration)
 * 4) *1 &mu;L 10X T4 DNA ligase buffer
 * 5) *6.5 &mu;L H2O
 * 6) *0.5 &mu;L T4 polynucleotide kinase
 * 7) Incubate at 37 &deg;C for 1 hr 30 mins
 * 8) Heat kill at 65 &deg;C for 20 mins
 * 9) Mix all 10&mu;L of kinase treated oligo with its pair to make duplex oligos.
 * 10) Heat to 70-75 &deg;C and cool to anneal oligo pairs.
 * 11) Mix all 20 &mu;L of each duplex together in one tube.
 * 12) Make up ligation reaction
 * 13) *1 &mu;L of the oligo mix
 * 14) *50 ng digested vector backbone
 * 15) **The assembled oligo construct should have complementary ends to the prepared vector
 * 16) *1 &mu;L 10X T4 DNA ligase buffer
 * 17) *H2O to 10 uL
 * 18) Add 0.5 &mu;L T4 DNA ligase
 * 19) Incubate for 1hr 30 mins at 16&deg;C.
 * 20) Transform.