Difference between revisions of "LongAmp PCR NEB"
From GcatWiki
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| − | !|''' | + | !|'''FINAL CONC.''' |
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|'''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 |
