Difference between revisions of "Q5 PCR NEB"

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• Do not exceed 50 µL final volume
 
• Do not exceed 50 µL final volume
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== Thermocycling Conditions==
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{| class="wikitable"
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!|'''STEP'''
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!|'''TEMPERATURE (°C)'''
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!colspan="2"|'''TIME'''
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|-
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|
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|
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|'''<6kb'''
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|'''≥6kb'''
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|-
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|'''Initial Denaturation'''
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|98
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|30 sec
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|1-3 min
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|-
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|'''30 Cycles''' ''denature''
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''anneal''
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''extend''
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|98
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50-71
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72
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|10 sec
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20 sec
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10 sec per kb
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|10 sec
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50 sec
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1 min per kb
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|-
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|'''Final Extension'''
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|72
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|2 min
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|2min
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|-
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|'''Hold'''
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|4-22
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|
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|
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|}
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To find annealing temperature, follow steps on the NEB Tm Calculator, [http://tmcalculator.neb.com/#!/] which should yield a temperature 3°C above the Tm of the lower primer

Revision as of 20:56, 14 June 2016

Reaction Setup

REAGENT VOLUME (µL) FINAL CONC.
10 µM Forward Primer 2.5 µL 0.5 µM
10 µM Reverse Primer 2.5 µL 0.5 µM
Template DNA Y (1ng) 1 ng
Q5 High-Fidelity 2X Master Mix 25 µL 1X
Nuclease-free Water 20- Y

Final Volume = 50µL


Notes:

• Q5 High-Fidelity 2X Master Mix has an error rate > 100-fold lower than that of Taq DNA Polymerase

• Q5 High-Fidelity 2X Master Mix can be stored at -20°C. If precipitate forms after thawing, resuspend before use

• Remember to first dilute primers in nuclease-free water if starting with a higher concentration

• Do not exceed 50 µL final volume

Thermocycling Conditions

STEP TEMPERATURE (°C) TIME
<6kb ≥6kb
Initial Denaturation 98 30 sec 1-3 min
30 Cycles denature

anneal

extend

98

50-71

72

10 sec

20 sec

10 sec per kb

10 sec

50 sec

1 min per kb

Final Extension 72 2 min 2min
Hold 4-22


To find annealing temperature, follow steps on the NEB Tm Calculator, [1] which should yield a temperature 3°C above the Tm of the lower primer

Retrieved from "https://gcat.davidson.edu/GcatWiki/index.php?title=Q5_PCR_NEB&oldid=18620"