Team:Caltech/Cloning

Two-Day Cloning System

We perfected an efficient 2-day cloning method for BioBricks to accomplish the large number of ligations we wanted. Constructs should be designed to accomplish as many ligations steps in parallel as possible.

Procedure

1. Day 1
   1. Miniprep cultures to extract DNA to be digested (if applicable).
   2. Digest (2.5hr) and ligate (1hr) appropriate DNA using Standard Assembly and (a) linearized backbone, (b) digested vector containing RFP or some other pigment gene, or (c) vector digested with Alkaline Phosphatase (CIP).
   3. Transform 2μL ligation product into desired cells via electroporation. Plate on LB-agar plates with appropriate antibiotic and grow overnight.
2. Day 2
   1. Perform CPCR on 2-10 colonies from each ligation plate to find clones with correct inserts. Depending on the backbone used, various fractions of the colonies may contain the incorrect insert, and will determine the number of colonies that must be tested. Make sure to make the parallel LB liquid cultures.
   2. CPCR product should be analyzed using standard agarose gel electrophoresis. Clones with the correct-sized inserts can be miniprepped the following morning for further cloning.
3. Day 3 (optional)
   1. Miniprepped samples can be sent out for sequencing to verify. This is only necessary when the construct is complete or to check progress on a very large construct.