Aug 08, 2022

Public workspaceLigation V.3

DOI
dx.doi.org/10.17504/protocols.io.5jyl89417v2w/v3
  • 1University of Wisconsin - Stout
  • Yeast ORFans CURE
Brian Teague
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DOI: dx.doi.org/10.17504/protocols.io.5jyl89417v2w/v3
Protocol Citation: Brian Teague 2022. Ligation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl89417v2w/v3Version created by Brian Teague
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 08, 2022
Last Modified: August 08, 2022
Protocol Integer ID: 68365
Abstract
A DNA ligase is an enzyme that forms phosphodiester bonds -- it can "glue" together two pieces of DNA. The ligase we're using comes from the T4 bacteriophage virus.

Why do we need to do a ligation anyway? Remember, we're going to use a Cas9 protein to "cut" your target gene in a yeast cell. We can program the Cas9 to cut a particular place using a "guide RNA." However, does Saccharomyces cerevisiae -- brewers' yeast -- make Cas9 or a guide RNA on its own? No! So we need to give the yeast cell a set of instructions for making the Cas9 protein and the guide RNA.

We do so by encoding those instructions on a plasmid -- a small circular piece of DNA -- and then putting that plasmid into the yeast cell. Then, the yeast cell "follows" the instructions to make the Cas9 protein and guide RNA. The plasmid we're making has several functional DNA sequences on it -- a diagram is below.



The functional sequences on the plasmid are denoted by colored boxes -- you can ignore most of them for now. Note, however, the boxes labelled Cas9 and Guide RNA -- these are the instructions for making Cas9 and the guide RNA. Remember, though, that each group is knocking out a different gene -- so each group needs a different plasmid!

The part that needs to be customized for each group is the "target sequence" in the above diagram. Your instructor started with a plasmid similar to the one above, but where the target sequence is instead replaced by gene for a green fluorescent protein (GFP) -- cells that express it turn bright green! They have cut out the GFP, and now you need to replace it with a targeting sequence for your gene, as in the diagram below.



One last thing. Where is your targeting sequence coming from? It's the annealed oligonucleotides from last time. You'll mix the plasmid backbone, your annealed oligos, the ligase enzyme, and a ligase buffer (which contains all of the chemicals the ligase needs to operate), and then the ligation will incubate at room temperature while the enzyme does its thing.
Image Attribution
By Madprime - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=2161789
Guidelines
This protocol involves pipetting very small quantities of liquid. Pipette carefully!
Materials
  • A 200 µl PCR tube
  • L2-01 backbone plasmid, 10 fmol/ul
  • Annealed oligonucleotides
  • ReagentT4 DNA LigaseNew England BiolabsCatalog #M0202
  • Reagent10X NEB T4 DNA ligase bufferNew England Biolabs
Note
The DNA ligase buffer is kept in single-use aliquots -- little tubes with an "L" on top. Take one for your group, use as much as you need, then discard the rest.


  • ReagentNuclease-free Water
  • ReagentTE Buffer


Protocol materials
Reagent10X NEB T4 DNA ligase bufferNew England Biolabs
ReagentNuclease-free Water
ReagentTE Buffer
ReagentT4 DNA LigaseNew England BiolabsCatalog #M0202
ReagentTE Buffer
ReagentNuclease-free Water
Reagent10X NEB T4 DNA ligase bufferNew England Biolabs
ReagentT4 DNA LigaseNew England BiolabsCatalog #M0202
Safety warnings
None of the materials in this lab are hazardous.

HOWEVER, we are shedding nucleases -- enzymes that degrade DNA -- all the time. Wear lab coats and gloves to keep your samples nuclease-free.
Before start
This protocol depends on the successful completion of the Annealing Oligonucleotides protocol, below:
Protocol
Annealing Oligonucleotides
NAME
Annealing Oligonucleotides
CREATED BY
Brian Teague

Ligation
Ligation
2h 30m
2h 30m
Dilute the annealed oligonucleotides in ReagentTE Buffer to make Amount100 µL of working stock at a final concentration of Concentration200 nanomolar (nM)

In the PCR tube, mix in order:
  • Amount4 µL ReagentNuclease-free Water
  • Amount2 µL of the L2-01 DNA plasmid backbone
  • Amount2 µL of the diluted annealed oligos
  • Amount1 µL Reagent10X NEB T4 DNA ligase bufferNew England Biolabs
  • Amount1 µL ReagentT4 DNA LigaseNew England BiolabsCatalog #M0202

Flick the tube several times to mix the components, then spin briefly in the microcentrifuge to collect everything at the bottom of the tube.
Incubate at room temperature at least Duration00:30:00 , and up to Duration02:00:00 (longer is better).

2h 30m
Store at Temperature-20 °C