Matt:LabNotes/2017-5-20: Difference between revisions

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Line 155: Line 155:
*Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
*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
*Discard supernatant and let dry in hood for 10min
*Resuspend each tube with 10ul and combine
*Resuspend each tube with 8ul and combine
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===Qubit Quantification===
===Qubit Quantification===
*10.3 ng/ul => 10.3 ng/ul / (157nt*325/nt + 79Da) = 201.5 nM (80ul)
*16.7 ng/ul => 16.7 ng/ul / (157nt*325/nt + 79Da) = 326.7 nM (80ul)
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Revision as of 21:47, 2 June 2017

Agi15k_Feb2017_V4 Probe Production

Notes from last time

  • Do 12 or 13 cycles Production PCR
  • Experiment with 1hr vs 2hr Lambda digestion
  • Think of way to maximize USER and DpnII digestion

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon Agi15k_Feb2017_V4 (25nM) 0.1 10
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4910
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold

EtOH Precipitation

  • 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 3,000rpm at 4C for 30 min
  • 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 5 min at 4C
  • Discarded supernatant and air-dried for 10 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification

  • 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: 244.2 ng/ul => ~97.64 ug

Lambda Exonuclease Digestion

  • 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
  • Incubate 2 tubes at 37C for 2hr and 2 tubes at 37C for 1hr
  • Purified with 8 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 1hr incubation: 72.3 ng/ul x 160ul = 11.6 ug
    • 2hr incubation: 81.7 ng/ul x 160ul = 13 ug
  • Conclusion: 2hr incubation is better

Remove Amplification Adapters

USER

  • Split into 6 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 52
USER 5
10X DpnII Buffer 8
H2O 15
Total 80
  • Incubate at 37C for 2.5 hours

DpnII

  • 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

  • Eluted 20ul each column (80ul total)
  • Nanodrop
    • 125.1ng/ul x 120ul = 15ug (% yield)

PAGE Size Selection

  • Run 4 gels
    • 200V for 40min
Components 4X Volume
V4 Probes 120
TBE-Urea Buffer 2X 120
Components 4X Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40

EtOH Precipitation

  • Put cut out gel in 8 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.
    • Had to use 2 more 1.5mL tubes to get all gel. Use 2.0mL tubes next time
  • Added 450 ul of 1X TE buffer to each
  • Vortexed for 120min 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
  • Precipitated in 10 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 10 tubes at -80C overnight
  • Spun 10 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 8ul and combine

Qubit Quantification

  • 16.7 ng/ul => 16.7 ng/ul / (157nt*325/nt + 79Da) = 326.7 nM (80ul)