Difference between revisions of "Q5 PCR NEB"

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(Created page with "== Reaction Setup == {| class="wikitable" !|'''REAGENT''' !|'''VOLUME (µL)''' !|'''FINAL CONC.''' |- |'''10 µM Forward Primer''' |2.5 µL |0.5 µM |- |'''10 µM Reverse Pr...")
 
(→‎Modifications)
 
(2 intermediate revisions by the same user not shown)
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• Do not exceed 50 µL final volume
 
• Do not exceed 50 µL final volume
 +
 +
== Thermocycling Conditions==
 +
 +
 +
{| class="wikitable"
 +
!|'''STEP'''
 +
!|'''TEMPERATURE (°C)'''
 +
!colspan="2"|'''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
 +
|2 min
 +
|-
 +
|'''Hold'''
 +
|4-22
 +
|
 +
|
 +
|}
 +
 +
 +
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
 +
 +
== Modifications ==
 +
If annealing temperature is above 72°C, use two-step PCR protocol below
 +
 +
 +
{| class="wikitable"
 +
!|'''STEP'''
 +
!|'''TEMPERATURE (°C)'''
 +
!colspan="2"|'''TIME'''
 +
|-
 +
|
 +
|
 +
|'''<6kb'''
 +
|'''≥6kb'''
 +
|-
 +
|'''Initial Denaturation'''
 +
|98
 +
|30 sec
 +
|1-3 min
 +
|-
 +
|'''30 Cycles''' ''denature''
 +
''anneal & extend''
 +
|98
 +
≥72
 +
|10 sec
 +
10 sec per kb
 +
|10 sec
 +
1 min per kb
 +
|-
 +
|'''Final Extension'''
 +
|72
 +
|2 min
 +
|2 min
 +
|-
 +
|'''Hold'''
 +
|4-22
 +
|
 +
|
 +
|}
 +
 +
Note that the length of each step should be thought of as a gradient, not a hard cutoff at 6kb.
 +
 +
For more information, visit the NEB Q5 High-Fidelity 2X Master Mix Protocol [https://www.neb.com/protocols/2012/08/29/protocol-for-q5-high-fidelity-2x-master-mix-m0492]

Latest revision as of 21:05, 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 2 min
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

Modifications

If annealing temperature is above 72°C, use two-step PCR protocol below


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

anneal & extend

98

≥72

10 sec

10 sec per kb

10 sec

1 min per kb

Final Extension 72 2 min 2 min
Hold 4-22

Note that the length of each step should be thought of as a gradient, not a hard cutoff at 6kb.

For more information, visit the NEB Q5 High-Fidelity 2X Master Mix Protocol [2]

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