Week 1 - 2015

Preparing the starting plasmid material

The first step is to obtain several micrograms of the two plasmids we will be working with, pKC7 and pUC18. 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. To isolate the plasmid DNA you will use the Thermoscientific™ GeneJET™ miniprep kit. Kits are designed to be easy to use, but you – the researcher – still must understand how the kit works. This means reading all the background material for the kit.

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 use these digests to check the plasmid quality and concentration. 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.

Before coming to lab this week you will need to

  • complete all readings listed below
  • complete Assignment #1 (see below)
  • prepare to use the protocols listed below by reading through them and making sure you understand what you will be doing
  • procure an appropriate lab notebook

You might also want to

Determine what the purpose of each of the steps in the purification protocol and where the plasmid DNA is at the end of each step.

Assignment #1 due at the start of the lab

  • Find the protocol for the GeneJET™ plasmid miniprep kit. Obtain protocol directly from Thermoscientific. The manual tends to get moved around but last time I looked it was near the bottom of the page, under the tab "resources". The manual should be about 10 pages long. The link was called "GeneJET Plasmid Miniprep Kit Product Information". Don't be afraid to click around a bit.
  • Print the GeneJET™ plasmid miniprep kit protocol and bring it to the lab. You only need to print the part of the manual that is the protocol. We will check that you have brought the correct protocol with you.
  • Make a list of the materials you will need to complete this weeks lab (you will hand in this list so make a second copy for yourself).
  • Bring an appropriate lab notebook (see the protocol "How to keep a notebook")

In your own words, briefly answer the following questions.

  1. In the GeneJET plasmid isolation protocol, where is the plasmid DNA located at the end of steps 4, 6, 7 and 10?
  2. If you are given a 1.5 ml tube of reagent and you have been told there is 10 µl in tube but you cannot see any liquid. What might have happened and how can you fix the problem?
  3. What is the purpose of the GeneJET column?

Read before coming to the lab

Safety
Introduction to the lab
Project overview - Step I - III.
Preparing for the lab
Molecular Techniques
About Plasmids
Plasmid and vector maps

all protocols you will be using - listed below

Protocols you will be using

Please carefully review all protocols before coming to lab - if any of the steps are not clear to you please ask before the lab (contact Lauri or post a comment below) or ask during the pre-lab).

  • How to keep a notebook – Bring an appropriate notebook to lab this week.
  • Proper use and care of pipetters
  • GeneJET™ plasmid miniprep kit. Obtain directly from Thermoscientific as described in assignment 1.

To do today:

Isolate plasmids

  • isolate pKC7 and pUC18 plasmid DNA using GeneJET™ plasmid miniprep kit
    • All buffers have been prepared as required (see Important Notes in the GeneJET protocol) and the cultures have been prepared for you as described in step 1 of Growth of Bacterial Cultures (both cultures were grown in LB medium supplemented with 100 µg/ml ampicillin).
    • You are starting at step 2 of Growth of Bacterial Cultures (this protocol is in the GeneJET manual and proceeds the plasmid isolation protocol).
    • For pUC18 use 3 ml of cells
    • For pKC7 use 8 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 HindIII; follow the manufactures suggested protocol. 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

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 and 15 µl of pKC7. Why?

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