Matt:LabNotes/2015-6-3

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CA12k_Nov2014_V4 Probe Production[edit]

Production PCR[edit]

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V4 (10nM) 0.2 20
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4900
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:20150603 CA12kNov2014 V4 ProductionPCR.JPG

QIAEX-II Purification[edit]

protocol

  • Divide into 8 5mL tubes of 1200ul each
  • Add 3600ul (3X volume) of Buffer QX1
  • Vortex the QIAEXII for 30sec
  • Add 30ul QIAEXII (30ul for every 10ug)
  • Incubate at 50C while mixing for 10min
    • Color should be yellow
  • Centrifuge sample for 30sec
  • Remove supernatant with a pipette
  • Wash the pellet with 500ul Buffer QX1
  • Resuspend the pellet by vortexing
  • Transfer to 1.5ml tubes
  • Centrifuge sample for 30sec and remove supernatant again with pipette
  • Wash the pellet twice with 500ul Buffer PE
  • Air-dry the pellet for 30min until the pellet turns white
  • To elute, resuspend with 20ul H2O and vortexing
  • Incubate at RT for 5min
  • Centrifuge for 30sec and pipette out supernatant with sample
  • Repeat 20ul H2O resuspension and incubation to improve yield 10-15%
  • Total volume: 300ul
  • Nanodrop quantification: 155.7 ng/ul -> 46.7 ug

Lambda Exonuclease Digestion[edit]

  • Divide into 6 pcr tubes of 50ul with total amplicon of <10ug each
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 1hr20min
  • Purified with 6 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 59.6ng/ul x 240ul = 14.3ug (61% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split into 4 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[edit]

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 2
100uM RE-DpnII_V4 guide oligo 5
H2O 8
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight (~15hrs)

Zymo Column Purification[edit]

  • Eluted 30ul each column (115ul total)
  • Nanodrop
    • 51.9ng/ul x 115ul = 6.2ug (43% yield)

PAGE Size Selection[edit]

  • Run 2 gels
    • 200V for 40min
Components 2X Volume
V4 Probes 115
TBE-Urea Buffer 2X 115
Components 2X Volume
Low Mass Ladder 2
TBE-Urea Buffer 2X 10
H2O 8
Total 20

File:2015-06-05 CA12kNov2014 V4 SizeSelect.jpg

EtOH Precipitation[edit]

  • Put cut out gel in 4 0.5mL tubes (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
  • Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
  • Added 450 ul of 1X TE buffer
  • Vortexed for >60min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transferred the clear spnt. to 4 Nanosep columns and centrifuged at 15,000 rpm for 3 min
  • Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  • Transferred spnt to 4 fresh 1.5 mL tube (~440ul per tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 4 tubes at -80C for overnight
  • Spun 4 tubes at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
  • Discard supernatant and let dry in hood for 10min
  • Resuspend each tube with 10ul and combine

Qubit ssDNA Quantification[edit]

21.4ng/ul => 21.4 ng/ul / (150nt*325/nt + 79Da) = 438nM (60ul)