Difference between revisions of "LongAmp PCR NEB"

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(Thermocycling Conditions)
(Reaction Setup:)
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{| class="wikitable"
 
{| class="wikitable"
!|'''STEP'''
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!|'''REAGENT'''
!|'''TEMPERATURE (°C)'''
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!|'''VOLUME (µL)'''
!|'''TIME'''
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!|'''FINAL CONC.'''
 
|-
 
|-
 
|'''10 µM Forward Primer'''
 
|'''10 µM Forward Primer'''

Revision as of 19:47, 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