Matt:LabNotes/2015-5-11: Difference between revisions

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[[File:2015-05-13_CA12kNov2014_V4_SizeSelect.jpg|450px]]


===EtOH Precipitation===
===EtOH Precipitation===

Latest revision as of 19:22, 21 May 2015

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:20150511 CA12kNov2014 V4 ProductionPCR.JPG

EtOH Precipitation[edit]

  • 8 5-ml tubes (with 12 wells of PCR product each) for V4
    • 1200ul PCR product
    • 3000ul 100% EtOH
    • 3ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • Centrifuged at 13000rpm at 4C for 20 min (in basement ultracentrifuge)
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 15 min at 4C
  • Discarded supernatant and air-dried for 10 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification[edit]

  • Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~400ul of V4 probes: 111.1 ng/ul => ~44.4 ug

Lambda Exonuclease Digestion[edit]

  • Divide into 4 pcr tubes of ~100ul with total amplicon of <10ug each
Components Volume
Amplicon 96
10X Lambda Exo Buffer 12
Lambda Exonuclease 12
Total 120
  • Incubated at 37C for 1hr
  • Purified with 4 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 92.9 ng/ul x 160ul = 14.9 ug (67% 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 20ul each column (80ul total)
  • Nanodrop
    • 103.7ng/ul x 80ul = 8.3ug (56% yield)

PAGE Size Selection[edit]

  • Run 2 gels
    • 200V for 40min
Components 2X Volume
V4 Probes 80
TBE-Urea Buffer 2X 80
Components 2X Volume
Low Mass Ladder 2
TBE-Urea Buffer 2X 10
H2O 8
Total 20

File:2015-05-13 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 to each
  • Vortexed for 60min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transferred the clear spnt. to 2 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 fresh 1.5 mL tube (~1700ul)
  • Combined with ~850ul from 5/5/2015
  • Precipitated in 6 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 6 tubes at -80C for 30min
  • Spun 6 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 Quantification[edit]

  • 22.5 ng/ul => 22.5 ng/ul / (150nt*325/nt + 79Da) = 461nM (60ul)