Q5 PCR NEB - Revision history

Anbuser: /* Modifications */

2016-06-14T21:05:58Z

‎Modifications

Anbuser: /* Thermocycling Conditions */

2016-06-14T21:03:17Z

‎Thermocycling Conditions

Anbuser: /* Reaction Setup */

2016-06-14T20:56:09Z

‎Reaction Setup

Anbuser: 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..."

2016-06-14T20:46:23Z

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..."

New page

== Reaction Setup ==

{| class="wikitable"
!|'''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

← Older revision		Revision as of 21:05, 14 June 2016
Line 126:		Line 126:
	\|		\|
	\|}		\|}
		+
		+	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]

@@ Line 77: / Line 77: @@
 |72
 |2 min
-|2min
+|2 min
 |-
 |'''Hold'''
@@ Line 87: / Line 87: @@
 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

← Older revision		Revision as of 20:56, 14 June 2016
Line 38:		Line 38:

	• 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
		+	\|2min
		+	\|-
		+	\|'''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