PCR Hood Rules!

These are the PCR Hood Rules in my lab that I have written up for 3rd Year Project Students. Applicable to similar labs.

G19 PCR HOOD RULES

The Cardinal Rule: NO PCR PRODUCTS TO BE EVER TAKEN INTO PCR HOODS

BE AWARE of the instructions for operating specific PCR Hoods
ALWAYS book the PCR Hood before use
ALWAYS wear fresh gloves when working at the PCR Hood. Keep a glove box handy in case you need to change gloves
NEVER wear a lab coat (used normally in this lab for post-PCR work only) to the PCR Hood
NEVER take the dedicated PCR pipettes or any other equipment including racks out of the PCR Hood.
NEVER place the icebox on a surface outside the PCR Hood. Wearing gloves, take the icebox in the PCR Hood to the ice machine, fill it up and return directly to the PCR Hood.
NEVER take storage boxes, etc. from the freezer into the PCR Hood. Take reagent and sample tubes out from storage boxes and place directly inside the PCR hood.
ALWAYS use tips, PCR tubes, 1.5ml tubes, microplates, and other plasticware dedicated for PCR use, taking them DIRECTLY from their bags. Most of these items are found in the store in front of G19.
NEVER put your hands, even when gloved, into bags containing PCR plasticware inside the PCR hood.
ALWAYS return your reagent to storage after each session
ALWAYS spin down your prepared PCR samples using the centrifuge and mix them using the plate mixer before putting them into the PCR machine

Taq:Pfu 20:1 Polymerase Mix (Taq 4.55Units/µl, Pfu 0.23Units/µl)

  1. Log and retrieve a fresh tube of Kapa Taq DNA Polymerase (5Units/µl, 50µl)
  2. Obtain a tube of Pfu polymerase (2.5Units/µl 50µl)
  3. To make a 20(Taq):1(Pfu) mix, add 5µl of Pfu to the Taq tube
  4. Label the Taq:Pfu tube with name, date and ‘Taq:Pfu 20:1’

PCR Water

  1. Wearing fresh gloves, obtain a Sterilin tube .
  2. Take the plastic bag-wrapped PCR clean Sigma water (18.2MΩ resistivity) to the PCR Hood and transfer the Sigma water bottle out of the plastic bag (which remains outside) directly into the PCR Hood.
  3. Add water to the Sterilin tube and re-wrap the Sigma water bottle outside the PCR Hood.
  4. Label the Sterilin tube with name, date and ‘PCR clean Sigma water’.
  5. After use, store the PCR clean water tube in the PCR clean cupboard.
  6. For secondary PCRs, you can make up a ‘Not PCR Clean’ water tube to keep on the bench as described here.

5mM Tris-HCl pH7.5 solution

  1. Add 1492µl PCR clean Sigma water (18.2MΩ resistivity) to a fresh 1.5ml Eppendorf tube
  2. Add 7.5µl of 1M Tris-HCl stock
  3. Label the tube ‘5mM Tris-HCL pH7.5

Use these if you want to file it or stick it up somewhere:

G19 PCR Hood Rules

G19 PCR Hood Rules2

Leave a Reply

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.