Week 3 - 2017

Gel-purify Gene A

NOTE – Each group needs to come in before the lab period to prepare an agarose gel. Please sign up, on UR Courses, for a time slot.


Last week we digested the vector pUC18 and the plasmid pKC7. We digested enough of each plasmid to set up a shotgun ligation and a targeted ligation. Before we can set up the targeted ligation, we need to separate the pKC7 fragment that contains Gene A from the plasmid fragment that does not contain gene A. We will do this by first separating the digested pKC7 fragments on a gel and then extracting the Gene A fragment from the agarose gel.

We have a lot of work to do in this week’s lab so please come to the lab ready to prepare and load the pKC7 digest on the gel.
Additionally, use your waiting times effectively by looking ahead to see what you can prepare while you are waiting.

Read/Watch/Do before coming to the lab

  • Prepare an agarose gel prior to the lab
    • The gel must be poured the day of your lab between 8:30 am and 2:00 pm. Please sign up for a time slot on UR Courses - it is best if you come as a group to make the gel, but if you can't get that to work, just one group member may come.
    • Make a 0.9% agarose in 30 ml 1X TAE with ethidium bromide added to a final concentration of 0.1 µg/ml
  • Using the "Quick guide to preparing and loading DNA samples" work out how you will prepare your pKC7 double digest for loading (we are loading the rest of the double digest from week 2).
  • Read Project overview – Step III - IV – Produce and prepare the DNA fragments for cloning & Ligation.
  • Prepare to use the protocols listed below.
  • Work out what you need to do to load the digested pKC7 plasmid. To keep things running on time, your group needs to be loading the gel within 10 min of arriving in the lab.

Protocols

  • General guidelines for molecular biology labs
  • Agarose gel electrophoresis of DNA
  • Estimating DNA concentration – agarose gel method
  • T4 DNA ligase information sheet, find this at Thermoscientific.
  • DNA extraction from agarose gel using QIAEX II Gel Extraction Kit - you will need to go to Qiagen, to obtain the protocol. Search for the name of the kit, look for the Handbook under the 'resources' tab. Note that you do not need to print the entire handbook - you should read it - but only print the protocol portion.

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

To do in lab today:

purify the Gene A fragment

  • prepare the double digested pKC7 for loading - load the entire digest
  • load the gel
    • no ladder needs to be run on this gel as we have already sized and quantified this sample
  • run the gel at 90 volts for 30 min
  • extract Gene A from the agarose gel – QIAEX II agarose gel extraction protocol
    • depending on what we decided when we quantified the pKC7 digest, you may need to reduce the elution volume – please check your previous notes and check with the lab instructor
    • in any case, DO NOT do a second elution step
    • use your waiting times to prepare for the next gel

Check the fragment's purity and concentration

  • prepare 1 µl of the purified Gene A for gel electrophoresis and load the second agarose gel with this sample – along with 5 µl of DNA ladder (use the same ladder as used in previous weeks)
  • run the second agarose gel at 90 volt for 40 min (we might need to run longer)
  • photograph the gel and estimate Gene A's concentration.

Is the purified fragment concentrated enough for ligation?

Look back in your notes to remind yourself how much purified Gene A we wanted to add to the ligation. Given the volume of the ligation that is left after adding the T4 DNA ligase, the ligase buffer and the digested pUC18, will you have enough room to add the amount of Gene A that we wanted to add?


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