Difference between revisions of "LongAmp PCR NEB"

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(Reaction Setup:)
(Modifications:)
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== Modifications: ==
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== Modifications ==
  
 
• If template is GC rich, a longer initial denaturation of 2-4 minute is recommended  
 
• If template is GC rich, a longer initial denaturation of 2-4 minute is recommended  

Revision as of 19:53, 14 June 2016

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]


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]