Matt:LabNotes/2016-9-14

From ZhangLabWiki
Revision as of 22:39, 19 September 2016 by >Mzcai (→‎Zymo Column Purification)
Jump to navigation Jump to search

CA12k_Nov2014 V6 (RevComp of V4) Probe Production

Production PCR

  • V6 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V6 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V6U (100uM) 0.4 20
AP2V6 (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

EtOH Precipitation

  • 12 5-ml tubes (with 8 wells of PCR product each)
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 1hr
  • 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 12 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 12 tubes of 100ul in 12 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
  • Measured concentration with Nanodrop:

~600ul of V6 probes: 113.2 ng/ul => ~ 67.9 ug

Lambda Exonuclease Digestion

  • Divide each into 8 pcr tubes of 75ul with total amplicon of <10ug each
Components Volume
Amplicon 75
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 5
Total 100
  • Incubated at 37C for 1hr
  • Purified with 8 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • V6: 80.3ng/ul x 320ul = 25.7ug (75.6% yield)

Remove Amplification Adapters

USER

  • Split each set into 5 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 63
USER 5
DpnII 10X 8
H2O 4
Total 80
  • Incubate at 37C for 3 hours

DpnII

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 2
100uM RE-DpnII_V6 guide oligo 5
H2O 8
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight (12 hrs)

Zymo Column Purification

  • Eluted 20ul each column (100ul total)
  • Nanodrop
    • V6: 118.9ng/ul x 100ul = 11.9ug

PAGE Size Selection

  • Run 4 gels for V6
    • 200V for 35min
Components 4X Volume
V6 Probes 100
H2O 20
TBE-Urea Buffer 2X 120
Components Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40