Aug 12, 2022

Public workspaceURA3 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. URA3 PCR. protocols.io https://protocols.io/view/ura3-pcr-ce6tthen
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 11, 2022
Last Modified: August 12, 2022
Protocol Integer ID: 68531
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 is for amplifying the URA3 gene from the YTK74 plasmid and adding the homology sequences needed for homology-directed repair.



Materials
  • YTK74 PCR template, 1 ng/ul
  • YFG-URA3 F forward primer, Concentration10 micromolar (µM) concentration
  • YFG-URA3 R reverse primer, Concentration10 micromolar (µM) concentration
  • ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
  • ReagentNuclease free waterContributed by users
  • A Amount200 µL PCR tube


Protocol materials
ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
Materials, Step 4
ReagentNuclease free water
Materials, Step 4
ReagentTE Buffer
Step 3
Safety warnings
None of the materials used 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.
Check that the thermocycler is programmed and holding at 98°C. The thermocycler program we're using is the following:


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


Note
Do not skip this step -- you don't want to wait for a thermocycler to warm up!

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 100 µl of each dilution.)

Note
Remember, by convention the blue-capped tubes from IDT 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 flick the tube several times, then spin down in a 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 98°C. Start the PCR program.
After the PCR program has run, store the tube at -20°C.