Difference between revisions of "Q5 PCR NEB"

Revision as of 21:03, 14 June 2016

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

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

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

@@ 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
+== 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
+sec per kb
+|10 sec
+min per kb
+|-
+|'''Final Extension'''
+|72
+|2 min
+|2 min
+|-
+|'''Hold'''
+|4-22
+|
+|
+|}