Matt:JustinLabNotes/2015-7-6

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Revision as of 18:17, 21 July 2015 by >Mzcai (→‎Lambda Exonuclease Digestion)
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CA12k_Nov2014_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V7 (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 700px

EtOH Precipitation

  • 12 5-ml tubes (with 8 wells of PCR product each) for V4
    • 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 12 tubes of 100ul in 12 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~618ul of V4 probes: 76.4 ng/ul => ~47.2 ug

Lambda Exonuclease Digestion

  • Divide into 6 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 6 Zymo columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 63.5ng/ul x 233ul = 14.8ug (63% yield)

Remove Amplification Adapters

USER

  • 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

  • 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 30ul each column (115ul total)
  • Nanodrop
    • 54.7ng/ul x 115ul = 6.3ug (43% yield)

PAGE Size Selection

  • Run 2 gels
    • 200V for 40min
Components 2X Volume
V4 Probes 120
TBE-Urea Buffer 2X 120
Components 3X Volume
Low Mass Ladder 2
TBE-Urea Buffer 2X 10
H2O 8
Total 20
  • Gel 1

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  • Gel 2

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EtOH Precipitation

  • Put cut out gel in 0.5mL tube (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 900 ul of 1X TE buffer (SHOULD HAVE BEEN 450ul)
  • Vortexed for overnight at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transferred the clear spnt. to 8 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 8 fresh 1.5 mL tube (~400ul per tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 8 tubes at -80C for 30min
  • Spun 8 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 5ul and combine

Qubit Quantification

  • 10.9ng/ul
    • 10.9 ng/ul / (150nt*325Da/nt + 79Da) = 239nM (38ul)