Week 1 - 2016

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.

Read/Watch/Do before coming to the lab

  • print and bring this guide and the guide for Week 2
  • complete the prelab survey on UR Courses - this is due at 4 pm on Friday, Sept. 9th.
  • watch the video "305 introductory screencast" - link on UR Courses
  • watch the video "Prelab screencast - week 1" - link on UR Courses
  • complete Assignment #1
  • complete prelab-1 quiz on UR Courses (due by 2:30 the day of your lab)
  • read the following
  • watch the video How to use pipettes
  • Find and read the full manual 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. Note that there may be a link to a one-page protocol; this protocol will not help you answer the quiz questions.
  • prepare to use the protocols listed below by reading through them and making sure you understand what you will be doing

Protocols you will be using

Please carefully review all protocols before coming to the lab; if any of the steps are not clear to you please ask 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
  • GeneJET™ plasmid miniprep protocol (note we are using the version for centrifuges) obtained directly from Thermoscientific as described above.

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 manufacturers 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
total vol 10 10

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 and 15 µl of pKC7. Why?

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