DOC

Laboratory 2 Manual (Version 2) - PCR (POLYMERASE CHAIN REACTION)

By Cindy Henry,2014-12-23 20:15
8 views 0
Laboratory 2 Manual (Version 2) - PCR (POLYMERASE CHAIN REACTION)

    BioE 498: Systems & Synthetic Biology

    Winter 2009

    Lab Manual

    Week 2

    BioE 498 Lab: In-Fusion BioBrick Assembly and Re-engineering

We will first use our purified PCR products to perform an In-Fusion PCR cloning reaction to fuse the two

    PCR products into a circular plasmid containing the functional genetic circuit. This In-Fusion reaction

    product will then be transformed into competent cells. We will perform colony PCR reactions to identify

    colonies that have the circuit of interest. Then we will grow overnight cultures from these colonies,

    perform minipreps to isolate the plasmids, and submit these plasmids for sequencing. Here’s the

    overview for the week:

    Monday In-Fusion PCR Cloning Reaction, Transformations

    Tuesday Transformant Colony Imaging, Colony PCR and Making Overnight Cultures

    Wednesday Colony PCR results, Making Glycerol Stocks, Minipreps, and DNA sequencing

I. IN-FUSION PCR CLONING REACTION

    Materials and Equipment

    In-Fusion PCR Cloning Kit

    Purified insert and vector PCR products (called “Insert” and “Vector” below)

    Molecular Grade Water

    Parafilm

    Non-shaking incubator (set to 37?C)

    Water bath (set to 50?C)

    Ice bucket with crushed ice

    TE Buffer (pH 8.0)

    Protocol

    1

    BioE 498: Systems & Synthetic Biology

    Winter 2009

    Lab Manual

    Week 2

    1. Write down the estimated sizes of the insert and vector PCR products by comparing the bands on the gel to the 1kb ladder bands. Do these match the predicted sizes of your insert and vector PCR products when looking at the primer sites on the template DNA? You do not need to answer this question now, but eventually you will want to see the Lab Introduction and APE files for more information on how to calculate the exact sizes of these PCR products. Then use these estimated PCR product sizes in the following formula to calculate the ng of insert DNA needed for the In-Fusion reaction. This formula calculates a 2:1 molar ratio of insert given 100 ng vector DNA.