Week 4

Gel-purify Gene A and set up ligations

NOTE – Each group needs to come in before the lab period to prepare an agarose gel (1 gel per group – details below).


In previous labs we prepared the vector (pUC18) for both ligations, and we prepared pKC7 for the shotgun ligation.
Today we will finish preparing the Gene A fragment for the targeted ligation by separating it from the rest of the pKC7 plasmid. 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. Finally, we will set up both the targeted and the shotgun ligation reactions.

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 before coming to the lab

Project overview – Step III - IV – Produce and prepare the DNA fragments for cloning & Ligation.

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.

To do today:

BEFORE THE LAB – each group needs to come in and prepare an agarose gel

* 0.9% agarose in 25 ml 1X TAE with ethidium bromide added to a final concentration of 0.1 µg/ml
* The gel must be poured the day of your lab between 8:30 am and 2:00 pm
* check back to the week we completed the digests to see how much digested pKC7 you will be loading
* work out how you will prepare your pKC7 digest for loading - see “Quick guide to preparing and loading sample” come prepared to set up and load your gel at 2:30!

purify the Gene A fragment

  • prepare the sample for loading
  • 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
    • while the gel is running determine the transformation efficiency of the competent cells you made last week - see “Preparation and transformation of competent bacteria: calcium chloride method”
  • 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 carry out step 11 (i.e. DO NOT do a second elution step)
    • use your waiting times to prepare for the next gel
  • 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)
    • While the gel is running fill in the ligation tables in your lab manual (as much as possible) so you are ready to set up the ligation as soon as you know the concentration of GeneA.
    • Get your instructor to check over your calculations.
  • photograph the gel and estimate the concentration of Gene A

set up the ligation reactions

  • T4 DNA ligase find this at Thermoscientific.
  • Set up 20 μl reactions.
  • The concentration of T4 DNA ligase that will be given to you will be 1 unit per μl.
  • We must see the completed ligation set-up before we will give you the components.
component amount of shotgun lig amount for targeted lig
vector (conc = )
digested pKC7 (conc = ) none
purified Gene A (conc = ) none
10X ligase buffer
T4 DNA ligase (conc = )
  • ligation reactions will be incubated at room temperature for 1 h and stored at -20 °C by your instructor

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