Matt:LabNotes/2015-1-13: Difference between revisions

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*Incubate at 37C for 1.5 hours
*Incubate at 37C for 2.5 hours
 
====DpnII====
====DpnII====
*Added 15ul of the following
*Added 15ul of the following

Revision as of 04:28, 14 January 2015

CA12k_Nov2014_V7 Probe Production

Production PCR

Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V7U (100uM) 0.4 20
AP2V7 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:011214 CA12kNov14 ProductionPCR V7.JPG

EtOH Precipitation

  • 6 15-ml tubes (with 8 wells of PCR product each) for V7
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • Centrifuged at 3000rpm at 4C for 30 min
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
  • Discarded supernatant and air-dried for 5 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification

  • Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~290ul of V7 probes: 93.2 ng/ul => ~27 ug

Lambda Exonuclease Digestion

  • Divide each into 3 pcr tubes of ~100ul with total amplicon of <10ug each
Components Volume
Amplicon 100
10X Lambda Exo Buffer 12.5
Lambda Exonuclease 12.5
Total 125
  • Incubated at 37C for 1hr
  • Purified with 3 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 62.9ng/ul x 120ul = 7.5ug (56% yield)

Remove Amplification Adapters

USER

  • Save 2ul for gel check later
  • Split into 2 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 59
USER 5
10X DpnII Buffer 8
H2O 8
Total 80
  • Incubate at 37C for 2.5 hours

DpnII

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 2
100uM RE-DpnII_V7 guide oligo 5
H2O 8
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight