AlanFung:LabNotes/Probe/2009-4-23: Difference between revisions

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>Alan6017518
(New page: ==Step 4: Adapter Removal== Exonuclease treatment '''Done on 4/22/09''' '''*Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated,...)
 
>Alan6017518
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==Step 4: Adapter Removal==
==Step 4: Adapter Removal==
Exonuclease treatment
Exonuclease treatment
'''Done on 4/22/09'''
'''Done on 4/22/09'''
'''*Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube  
 
 
*'''Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube  
*Add in (1/10 of total volume) 6uL 10X Exo buffer  
*Add in (1/10 of total volume) 6uL 10X Exo buffer  
*Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR'''
*Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR'''

Revision as of 17:28, 23 April 2009

Step 4: Adapter Removal

Exonuclease treatment


Done on 4/22/09


  • Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube
  • Add in (1/10 of total volume) 6uL 10X Exo buffer
  • Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR

Purify the reaction with Quaquick columns

  • Add 5X sample volume of PB buffer into each tube, mix well
  • Load mixture onto column
  • Centrifuge at 14,000rpm for 1min
  • Discard flow through and add 750uL PE buffer to each column
  • Repeat Centrifuge
  • Discard flow through
  • Repeat centrifuge
  • Transfer columns to clean 1.5mL tubes
  • Place the column/tube assemblies on bench top wait for 2min
  • Add in 50uL ddH2O to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min
  • Repeat centrifuge
  • Measure concentration with Nanodrop

Incubation with USER enzyme:

  • Add 5ul DpNII 10x buffer and 2 uL of USER (1K/ml)enzyme incubate for 2 hours on 37C.