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Golden Gate for pKL2's

Updated 6/5/2018 07:39 PM
Step-by-step
by Boston University iGEM WL 2018 (created by Cassandra-Lynn Leach)

Introduction

pKL2 assembly!

Materials

Procedure

  1. Mix your pKL1's, water, and entry vector for a total of 8µL. You want 150ng of each pKL1 and 20-30 of pKLE.
  1. Add 1µL of T4 DNA ligase buffer and .5µL of BsmBI enzyme
  1. Put in 42 degree water bath for at least 30 min (can stay for an hour or hour and a half)
  1. Add .5µL of T7 ligase and and put in thermocycler on golden 2
  1. Put the assembly in the thermocycler on setting golden2. This should take ~3 hours.

The cycle is as follows:

1) 16oC for 30 min.

2) 42oC for 2 min.

3) 16oC for 2 min.

4) Repeat 2 and 3 for 20 cycles.

Comments

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