LongAmp PCR NEB

Reaction Setup

REAGENT	VOLUME (µL)	FINAL CONC.
10 µM Forward Primer	2 µL	0.4 µM
10 µM Reverse Primer	2 µL	0.4 µM
Template DNA	Y (1ng)	1 ng
LongAmp Taq 2X Master Mix	25 µL	1X
Nuclease-free Water	21- Y

Final Volume = 50µL

Notes:

• LongAmp Taq 2X Master Mix can be used to amplify templates of up to 30kb long.

• LongAmp Taq 2X Master Mix can be stored at -20°C, however, it is only stable for 15 freeze-thaw cycles. For more frequent use, NEB recommends storing it at 4°C.

• 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
Initial Denaturation	94	1 min
30 Cycles denature anneal extend	94 45-60 65	20 sec 40 sec 50 sec per kb
Final Extension	65	10 min
Hold	4-22

To find annealing temperature, follow steps on the NEB Tm Calculator, [1] which should yield a temperature 5°C below the Tm of the lower primer

Modifications

• If template is GC rich, a longer initial denaturation of 2-4 minute is recommended

• If annealing temperature is above 60°C, use two-step PCR protocol below

STEP	TEMPERATURE (°C)	TIME
Initial Denaturation	94	1 min
30 Cycles denature anneal & extend	94 60-65	20 sec 50 sec per kb
Final Extension	65	10 min
Hold	4-22

For more information, visit the NEB LongAmp Protocol [2]