Muratore:Protocols/PCR/Basic

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Current revision (17:06, 8 June 2011) (view source)
 
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==Basic PCR protocol==
 
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'''Basic PCR protocol'''
'''Basic PCR protocol'''
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!Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!5'primer !!3'primer !!dNTPs !!Pfu Turbo !!wax
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!Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!5'primer !!3'primer !![[dNTPs]] !![[Pfu Turbo]] !!wax
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! !! !!(10X) !!(10 μg/mL) !!(4 μM) !!(4 μM) !!(10 mM ea) !!(2.5 U/μL) !!
! !! !!(10X) !!(10 μg/mL) !!(4 μM) !!(4 μM) !!(10 mM ea) !!(2.5 U/μL) !!

Current revision

Basic PCR protocol
Tube sterile H2O Pfu Buffer Template 5'primer 3'primer dNTPs Pfu Turbo wax
(10X) (10 μg/mL) (4 μM) (4 μM) (10 mM ea) (2.5 U/μL)
Experimental 72 μL 10 μL 5 μL 5 μL 5 μL 2 μL 1 μL 50 μL
(-) Control 73 μL 10 μL 5 μL 5 μL 5 μL 2 μL 0 μL 50 μL
  • Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
  1. 5' @ 95°C
  2. 30" @ 95°C
  3. 2' @ Tm-5°C
  4. 2' @ 78°C
  5. repeat steps 2-4 x 29 more times
  6. 5' @ 78°C
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