AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol

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Current revision (12:29, 15 October 2012) (view source)
(Protocol)
 
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http://stanxterm.aecom.yu.edu/wiki/data/Product_manuals_attach/pfuturbo.pdf PCR manual
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[http://stanxterm.aecom.yu.edu/wiki/data/Product_manuals_attach/pfuturbo.pdf PCR manual]
==Materials==
==Materials==
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temperature cycler
temperature cycler
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==Directions==
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==Parameters==
*for DNA vectors (plasmids) of less than 10 kbases)
*for DNA vectors (plasmids) of less than 10 kbases)
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**The primers must each be at 100-200 nh/mL
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**The primers must each be at 100-200 ng/μL
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** dNTPS 100-250 μM of each dNTPs
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** 100-250 μM of each dNTPs
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** denaturing temperature is 95 °C
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**denaturing temperature is 95 °C
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** extension temperature is 72 °C (extension time 1 min per kb)
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**extension temperature is 72 °C (extension time 1 min per kb)
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** 25-30 cycles
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**25-30 cycles
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** enzyme concentration is 2.5 units of enxyme/ 50 μL of reaction for vector targets up to 19 kb and must be hte last component added to PCR mixture (ie after dNTPS)
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** enzyme concentration is 2.5 units of enzyme/ 50 μL of reaction for vector targets up to 19 kb and must be the last component added to PCR mixture (ie after dNTPS) to prevent degradation of the primers.
==Protocol==
==Protocol==
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# prepare the reaction mixture of 50 μL
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*prepare the reaction mixture of 50 μL
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##add the components in order
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**add the components in order
Component Amount per reaction
Component Amount per reaction
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component Amount per reaction'''
| align="center" style="background:#f0f0f0;"|'''Component Amount per reaction'''
|-
|-
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| Distilled water (dH2O) 40.6 μl
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| Distilled water (dH2O)           40.6 μl
|-
|-
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| 10× cloned Pfu reaction buffer 5.0 μl
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| 10× cloned Pfu reaction buffer   5.0 μl
|-
|-
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| dNTPs (25 mM each dNTP) 0.4 μl
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| dNTPs (25 mM each dNTP)           0.4 μl
|-
|-
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| DNA template (100 ng/μl) 1.0 μl
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| DNA template (100 ng/μl)         1.0 μl
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|-
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| Primer #1 (100 ng/μl) 1.0 μl
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| Primer #1 (100 ng/μl)             1.0 μl
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|-
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| Primer #2 (100 ng/μl) 1.0 μl
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| Primer #2 (100 ng/μl)             1.0 μl
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|-
| PfuTurbo® DNA polymerase (2.5 U/μl) 1.0 μl (2.5 U)
| PfuTurbo® DNA polymerase (2.5 U/μl) 1.0 μl (2.5 U)
|-
|-
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| Total reaction volume 50 μl
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| Total reaction volume             50 μl
|}
|}
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#immediately before thermal cycling, aliquot 50 μl of the reaction mixture into a sterile 0.5 mL micro centrifuge tube
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*immediately before thermal cycling, aliquot 50 μl of the reaction mixture into a sterile 0.5 mL micro centrifuge tube
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*thermocycle
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#thermocycle
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**for targets less than 10 kb vector DNA
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## for targets less than 10 kb vector DNA
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1. 2 min at 95 °C <br>
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1. 2 min at 95 °C
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2. repeat this 30 times<br>
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2. repeat this 30 times
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     a) 30 s at 95 °C <br>
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     a) 30 s at 95 °C  
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     b) 30 s at Tm - 5 °C <br>
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     b) 30 s at Tm - 5 °C  
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     c) 1 min at 72 oC for each 1 kb<br>
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     c) 1 min at 72 oC for each 1 kb
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3.  10 min at 72 °C<br>
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3.  10 min at 72 °C
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4. set temperature to 0 °C<br>
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4. set temperature to 0 °C
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==Notes==
==Notes==
* Backbone size w/o insert (bp):7328 bp for ADA
* Backbone size w/o insert (bp):7328 bp for ADA

Current revision

PCR manual

Contents

Materials

  • primers
  • plasmid
  • dexynucleoside triphosphate mixture (dNTPs)

Equipment

PCR tubes temperature cycler

Parameters

  • for DNA vectors (plasmids) of less than 10 kbases)
    • The primers must each be at 100-200 ng/μL
    • 100-250 μM of each dNTPs
    • denaturing temperature is 95 °C
    • extension temperature is 72 °C (extension time 1 min per kb)
    • 25-30 cycles
    • enzyme concentration is 2.5 units of enzyme/ 50 μL of reaction for vector targets up to 19 kb and must be the last component added to PCR mixture (ie after dNTPS) to prevent degradation of the primers.

Protocol

  • prepare the reaction mixture of 50 μL
    • add the components in order

Component Amount per reaction

Component Amount per reaction
Distilled water (dH2O) 40.6 μl
10× cloned Pfu reaction buffer 5.0 μl
dNTPs (25 mM each dNTP) 0.4 μl
DNA template (100 ng/μl) 1.0 μl
Primer #1 (100 ng/μl) 1.0 μl
Primer #2 (100 ng/μl) 1.0 μl
PfuTurbo® DNA polymerase (2.5 U/μl) 1.0 μl (2.5 U)
Total reaction volume 50 μl
  • immediately before thermal cycling, aliquot 50 μl of the reaction mixture into a sterile 0.5 mL micro centrifuge tube
  • thermocycle
    • for targets less than 10 kb vector DNA

1. 2 min at 95 °C
2. repeat this 30 times

   a) 30 s at 95 °C 
b) 30 s at Tm - 5 °C
c) 1 min at 72 oC for each 1 kb

3. 10 min at 72 °C
4. set temperature to 0 °C

Notes

  • Backbone size w/o insert (bp):7328 bp for ADA
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