Aug 15, 2022

Public workspaceKnockout PCR

This protocol is a draft, published without a DOI.
  • 1University of Wisconsin - Stout
Brian Teague
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Protocol Citation: Brian Teague 2022. Knockout PCR. protocols.io https://protocols.io/view/knockout-pcr-cfamtic6
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 15, 2022
Last Modified: August 15, 2022
Protocol Integer ID: 68653
Keywords: genomic, pcr, knockout
Abstract
The polymerase chaine reaction (PCR) amplifies linear DNA using a DNA polymerase enzyme and a pair of short single-stranded DNA "primers." This protocol amplifies a region that spans the Cas9 cut site so you can verify whether or not the URA3 "patch" inserted correctly (and thus disabled your gene.)




Materials
  • ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
  • Yeast genomic DNA
  • "YFG KO F" Forward primer, 10 µM concentration
  • "YFG KO R" Reverse primer, 10 µM concentration
  • ReagentNuclease free waterContributed by users
  • ReagentTE BufferContributed by users


Protocol materials
ReagentTE Buffer
Materials, Step 3
ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
Materials, Step 4
ReagentNuclease free water
Materials, Step 4
Safety warnings
None of the materials in this protocol are particularly hazardous. HOWEVER, we are shedding nucleases -- enzymes that degrade DNA -- all the time. Wear lab coats and gloves to keep your samples nuclease-free.
Check that the thermocycler is programmed with the following program and holding at Temperature98 °C



AB
98° for 30 seconds
Repeat 35 times:
98° for 5 seconds
60° for 15 seconds
72° for 1 minute
72° for 2 minutes
Hold at 8°


Note
Do not skip this step!


Grab an ice bucket and fill it with ice. If you don't have an ice bucket, a beaker will work in a pinch.
If necessary, dilute the primers to a concentration of Concentration10 micromolar (µM) in ReagentTE BufferContributed by users . (Make Amount100 µL of each dilution.)

Note
Remember, the blue-capped tubes have a concentration of Concentration100 micromolar (µM) .


Mix the following in a PCR tube on ice, in this order:
  • Amount7 µL ReagentNuclease free waterContributed by users
  • Amount1 µL forward primer
  • Amount1 µL reverse primer
  • Amount1 µL template DNA
  • Amount10 µL ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S





Mix the reaction by gently flicking the tube several times, then spin down in the microfuge.

Note
Do this quickly and return the tube to the ice bucket ASAP. There aren't enough thermocycler blocks for every group -- you may need to wait to share a thermocycler with other groups.

Transfer the tube from ice to a pre-heated thermocycler holding at Temperature98 °C . Start the PCR program.

After the PCR program has run, store the tube atTemperature-20 °C