Week 3 - 2015

Gel-purify Gene A

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


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 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 30 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

Check purity and determine concentration of the purified Gene A fragment

  • 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 the concentration of Gene A

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