User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/08/15: Difference between revisions

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(Autocreate 2012/08/15 Entry for User:Tamra_L._Fisher/Notebook/Research_for_Dr._Hartings)
 
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==Entry title==
==Amplification of Last Night's Ligation Product==
* Insert content here...
This procedure was done for the wild-type Asc Hb that was ligated with pQE-80-L-Kan last night. The pQE-80 primer was used:
# Mix 29μL of Nuclease-free water, 10μL of 5X Phusion HF Buffer, 1μL of 10mM dNTPs, 2.5μL 10μM forward primer, 2.5μL 10μM reverse primer, and 5.5μL of the ligation product.
# Add 0.5μL of Phusion DNA Polymerase, mix the solution, and centrifuge shortly.
# Put 50μL of wax on top of the mixture and place on the pre-heated block of the thermocycler.
# Start with 30 seconds at 98°C on the thermocycler.
# Cycle through the following 40 times:
#* 10 seconds at 98°C
#* 30 seconds at 62°C
#* 2.5 minutes at 72°C
# A final extension step of 10 min was done at 72°C.
# The thermocycler was then held at 4°C.
 
Most components for this PCR came from the [http://www.neb.com/nebecomm/ManualFiles/manualE0553.pdf  Phusion® High-Fidelity PCR Kit].




Revision as of 07:00, 15 August 2012

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Amplification of Last Night's Ligation Product

This procedure was done for the wild-type Asc Hb that was ligated with pQE-80-L-Kan last night. The pQE-80 primer was used:

  1. Mix 29μL of Nuclease-free water, 10μL of 5X Phusion HF Buffer, 1μL of 10mM dNTPs, 2.5μL 10μM forward primer, 2.5μL 10μM reverse primer, and 5.5μL of the ligation product.
  2. Add 0.5μL of Phusion DNA Polymerase, mix the solution, and centrifuge shortly.
  3. Put 50μL of wax on top of the mixture and place on the pre-heated block of the thermocycler.
  4. Start with 30 seconds at 98°C on the thermocycler.
  5. Cycle through the following 40 times:
    • 10 seconds at 98°C
    • 30 seconds at 62°C
    • 2.5 minutes at 72°C
  6. A final extension step of 10 min was done at 72°C.
  7. The thermocycler was then held at 4°C.

Most components for this PCR came from the Phusion® High-Fidelity PCR Kit.



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