Week 1 - 2017

Preparing the starting plasmid material

The first step is to obtain several micrograms of the two plasmids we will be working with, Plasmid Maps. The easiest way to obtain plasmids is to let bacteria make them for us. Once the bacteria have produced the plasmids we simply isolate the plasmids from the rest of the bacterial contents. See Plasmids and Vectors for more about plasmids.

Both plasmids are harboured in Escherichia coli. We will use alkaline-lysis to isolate the plasmid DNA and a silica column to purify the DNA. Most research labs will use prepared kits to isolate plasmid DNA. These kits use a lysis and clean up protocol that is similar to what we are using but we save a considerable amount of money by preparing our own solutions and just purchasing the silica columns. A kit for 250 plasmid preps costs about $300; buying the columns costs about $100 and preparing the solutions costs about $50.

A kit is a commercially prepared set of components, packaged together for use in a specific protocol.

After isolating the plasmids you will set up restriction digests of each plasmid. Next week you will determine the plasmid quality and concentration by analyzing the resulting fragments on by agarose gel electrophoresis. For this week you don't need to worry about how the digest works, we will talk about that more in next week's lab.

Read/Watch/Do before coming to the lab

Protocols you will be using

Please carefully review all protocols before coming to the lab; if any of the steps are not clear, ask for clarification before the lab (contact Lauri or post a question on the UR Courses Forum) or ask during the pre-lab). * prepare to use the protocols listed below by reading through them and making sure you understand what you will be doing

  • How to keep a notebook – Bring an appropriate notebook to lab this week.
  • Proper use and care of pipetters
  • Alkaline lysis & plasmid purification using EconoSpin (in your protocol book).

To do today:

Isolate plasmids

  • isolate pKC7 and pUC18 plasmid DNA
    • All buffers have been prepared as required and the cultures have been prepared for you as described in the plasmid isolation protocol (both cultures were grown in LB medium supplemented with 100 µg/ml ampicillin).
    • You are starting at step 1.
    • For pUC18 use 3 ml 1.5 mL of cells
    • For pKC7 use 5 ml of cells
  • after setting up the digests, isolated plasmids will be collected and stored at -20 °C

Set up restriction digests

Set up a double digest with the FastDigest restriction enzyme Hind III; follow the manufacturer's (ThermoFisher) suggested protocol (we will provide the details in lab). You will need to calculate the appropriate volumes of each component and fill it in the table below. We will check your table for accuracy before you set up the reaction.

  • Plasmid and enzyme volumes are given
  • Final buffer concentration is 1X for both enzymes.
  • Final digest volume for both is 10 µl (make up the difference using H20)
  • Incubate digests as recommended by the enzyme manufacturer

NOTE: all volumes are in µl

component pKC7 digest pUC18 digest
DNA 5 2
HindIII 1 1
10 X FastDigest buffer
H20
total vol 10 10

Prepare for gel loading next week

Your first task next week will be to load the digests on an agarose gel. To speed up the lab next week, we will add the dye this week and make notes on how to load the gel.

  • Using the "Quick guide to preparing and loading DNA samples", determine how much dye you need to add to each digest in preparation.
  • When incubation is complete, add the loading dye.
  • Indicate in your lab notebook how you will load the gel next week (e.g. gel loading order and volumes). Two groups will share a gel - we will give you the specifics in lab.

Further Questions

Answers do not need to be submitted.

  1. Both plasmids are being digested in a total volume of 20 µl, but we are digesting 2.5 times more pKC7 than pUC18. Why?

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