Difference between revisions of "LongAmp PCR NEB"
From GcatWiki
(→Reaction Setup:) |
(→Thermocycling Conditions) |
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| Line 59: | Line 59: | ||
''extend'' | ''extend'' | ||
|94 | |94 | ||
| − | 45-60 | + | 45-60 |
65 | 65 | ||
| Line 65: | Line 65: | ||
40 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 [http://tmcalculator.neb.com/#!/] | ||
| + | |||
| + | |||
| + | == 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 | ||
| + | |||
| + | {| class="wikitable" | ||
| + | !|'''STEP''' | ||
| + | !|'''TEMPERATURE (°C)''' | ||
| + | !|'''TIME''' | ||
| + | |- | ||
| + | |'''Initial Denaturation''' | ||
| + | |94 | ||
| + | |1 min | ||
| + | |- | ||
| + | |'''30 Cycles''' ''denature'' | ||
| + | ''anneal & extend'' | ||
| + | |94 | ||
| + | 60-65 | ||
| + | |20 sec | ||
50 sec per kb | 50 sec per kb | ||
|- | |- | ||
Revision as of 19:51, 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 |
