Difference between revisions of "PCR Amplification of DNA"

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(Created page with '==Materials== *Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined *dNTPs – dil...')
 
 
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==SOP==
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*[[SOP - Electrophoresis]]
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==Materials==
 
==Materials==
*Primers – order from IDT prediluted to 100 mM in TE.  Make working solution of 1 uM (100x) in water with both sense and antisense primers combined
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*Primers – order from IDT prediluted to 100 mM in TE.  Make working solution of 0.4uM-1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)
*dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL)
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*DreamTaq Green PCR Master mix - contains dNTPs, polymerase, salts, and buffer with loading dye  https://www.thermofisher.com/order/catalog/product/K1081?ICID=search-product#/K1081?ICID=search-product
*Template – generally 1uL or less of a plasmid miniprep
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*RNAse-Free water - comes in DreamTaq kit
*Polymerase – use Pfu Turbo for cloning and Taq for noncloning.  Use appropriate buffer.
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==Protocol==
 
==Protocol==
#Use the following volumes per reaction
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*Use the following volumes per reaction
*Buffer, 5 uL of 10X buffer
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::*12.5 uL DreamTaq
*Primers, 10uL of 1uM stock solution in water (both primers combined)
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::*7.5 uL RNAse-Free water
*dNTPs, 5uL of 2 mM
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::*5.0 uL Working stock Primer (of 0.4-1uM stock solution in water (both primers combined))
*Sterile water, 28 uL
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*Template 1 uL
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==PCR Program==
*Polymerase 1 uL
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*For animals: use programs denoted in [[Genotyping Program]]
  
#Run PCR Program.  Normally use touchdown PCR ('''DAVETD''') as follows:
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*Run PCR Program (approx 3.5 to 4 hours).  Normally use touchdown PCR ('''DAVETD''') as follows:
##1 min at 94
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:#1 min at 94
##30s at 65
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:#30s at 65
##2 min/kb at 72
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:#2 min/kb at 72
##30s at 94
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:#30s at 94
##30s at 63 then -0.5/cycle
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:#30s at 63 then -0.5/cycle
##2 min/kb at 72
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:#2 min/kb at 72
##Repeat steps 4-6 28 times
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:#Repeat steps 4-6 28 times
##30s at 94
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:#30s at 94
##30s at 45
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:#30s at 45
##11 min at 72
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:#11 min at 72
##hold at 4 until ready
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:#Hold at 4 until ready
#Purify PCR product if necessary using Qiagen kit (Add 5x PB)
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*Purify PCR product if necessary using Qiagen kit (Add 5x PB)

Latest revision as of 16:57, 8 June 2020

SOP

Materials

Protocol

  • Use the following volumes per reaction
  • 12.5 uL DreamTaq
  • 7.5 uL RNAse-Free water
  • 5.0 uL Working stock Primer (of 0.4-1uM stock solution in water (both primers combined))

PCR Program

  • Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR (DAVETD) as follows:
  1. 1 min at 94
  2. 30s at 65
  3. 2 min/kb at 72
  4. 30s at 94
  5. 30s at 63 then -0.5/cycle
  6. 2 min/kb at 72
  7. Repeat steps 4-6 28 times
  8. 30s at 94
  9. 30s at 45
  10. 11 min at 72
  11. Hold at 4 until ready
  • Purify PCR product if necessary using Qiagen kit (Add 5x PB)