Nov 25, 2020

Public workspaceFeacalis_colony_PCR

This protocol is a draft, published without a DOI.
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Protocol Citation: Elizabeth Fozo 2020. Feacalis_colony_PCR. protocols.io https://protocols.io/view/feacalis-colony-pcr-bp4pmqvn
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: November 25, 2020
Last Modified: November 25, 2020
Protocol Integer ID: 44911
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Abstract
E. faecalis colony PCR using taq polymerase
E. faecalis colony PCR using taq polymerase
E. faecalis colony PCR using taq polymerase
Transfer 1 colony, depending on size to 50-100µLwater. Normal size colonies can go in ~65µLwater.
To a PCR tube add:
  • 15 µL colony water*
  • 1.25 µL primer 1 (20 µM stock)
  • 1.25 µL primer 2 (20 µM stock)
  • 2.5 µL10X thermopolbuffer
  • 0.5 µL10 mM dNTPs
  • 0.5 µL Taq polymerase
  • 4 µL water
Total = 25 µL
Note
*Master mix can be made with all ingredients except the colony water for multiple reactions using the same primers.

Use the following cycle in the thermocycler:
94°C        10 min 1 cycle

94°C         15 sec
  
54°C         30sec(5°C below Tm)

72°C         1 min/kb     25-30 cycles

72°C         10 min        1 cycle

15°C          HOLD