Aug 17, 2022

Public workspacePCR (Instructor Protocol)

This protocol is a draft, published without a DOI.
  • 1University of Wisconsin - Stout
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Protocol Citation: Brian Teague 2022. PCR (Instructor Protocol). protocols.io https://protocols.io/view/pcr-instructor-protocol-cfaptidn
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 17, 2022
Protocol Integer ID: 68655
Keywords: pcr
Abstract
This is the instructor protocol for the CURE's two PCRs,
Protocol
URA3 PCR
NAME
URA3 PCR
CREATED BY
Brian Teague

and
Protocol
Knockout PCR
NAME
Knockout PCR
CREATED BY
Brian Teague

The URA3 PCR is preparative, to make the URA3 cassette for the gene knockout. The knockout PCR is diagnostic, to see whether the knockout succeeded.
Materials
  • ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
  • E. coli harboring the YTK74 plasmid
  • ReagentMonarch® Plasmid Miniprep Kit New England BiolabsCatalog #T1010
  • ReagentNuclease free water

Protocol materials
ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
Materials, Step 1
ReagentMonarch® Plasmid Miniprep Kit New England BiolabsCatalog #T1010
In Materials, Safety warnings, Step 3
ReagentNuclease free water
Materials, Step 2
Safety warnings
The solutions in the ReagentMonarch® Plasmid Miniprep Kit New England BiolabsCatalog #T1010 have safety implications (particularly the lysis solution, which is quite caustic.) Wear appropriate PPE.

Setup
Setup
Aliquot ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S into Amount100 µL aliquots, 1 per 4 students.

If not done already, aliquot ReagentNuclease free waterContributed by users into Amount1 mL aliquots, 1 per 4 students.

For the URA3 PCR: grow and miniprep the E. coli hosting YTK74, using ReagentMonarch® Plasmid Miniprep Kit New England BiolabsCatalog #T1010 or similar. Dilute to 1 ng/ul in elution buffer and make Amount20 µL aliquots, 1 tube for every 4 students.

Instructor Tips & Common Student Errors
Instructor Tips & Common Student Errors
Instructor Tips
  • Students often struggle with PCR. It's tetchy - I still struggle with it occasionally! Several choices make success more likely:
- Hot-start Q5 master mix. The Q5 master mix is really robust, and the hot-start formulation keeps the enzyme from chewing up primers in the wait between mixing the reaction and starting it in the thermocycler.
- For the URA3 preparative PCR - purified plasmid template. No nuclease contamination from suboptimal genomic DNA preps.

  • The idea that PCR works by an exponential DNA amplification is another thing that students often struggle with. I firmly believe that students should understand why they're doing what they're doing instead of just following a protocol. How best to motivate and evaluate student understanding is something I'm still struggling with.

  • Related to the above, the fact that there are three sets of oligos -- and that they're used in different ways, for different protocols -- is another thing to help students with. Why can't you just reuse the targeting oligos for PCR?

  • There is a LOT of pipetting of small volumes here. A reminder of good small-volume pipetting technique would not go amiss.

  • Interpreting the PCR gel often helps students solidify their understanding of what's going on here. Why is it important to estimate the PCR amplicon's size, not just see whether there's DNA there or not? For the knockout PCR, what does a long amplicon vs a short amplicon tell us?

Common student errors
  • Used wrong primers or template
  • Pipetting errors!