Matt:LabNotes/2016-9-14

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Revision as of 01:02, 17 September 2016 by >Mzcai (→‎Lambda Exonuclease Digestion)
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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 64
USER 5
Total 80
  • Incubate at 37C for 3 hours

DpnII

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 10
100uM RE-DpnII guide oligo 5
 RE-DpnII guide oligo:
 V6: RE DpnII V6
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight (16 hrs)

TBU Gel: Digestion Check

  • Combine into 1 tube for each set
  • 2ul Digested Sample + 2ul 2X Urea Loading Dye
  • 5ul Undigested sample (10nM V8 1st round amplicon) + 5ul 2X Urea Loading Dye
  • 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye

[[File:]]

Zymo Column Purification

  • 3 columns for each set
  • Eluted 20ul each column (60ul total)
  • Nanodrop
    • V6: 137.3ng/ul x 60ul = 8.2ug

PAGE Size Selection

  • Run 4 gels for V6
    • 200V for 35min
Components 2X Volume
V6 Probes 60
H2O 60
TBE-Urea Buffer 2X 120
Components Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40

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.
  • Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
  • Added 900 ul of 1X TE buffer
  • Vortexed for 60 min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transfered the clear spnt. to 8 Nanosep column and centrifuged at 15,000 rpm for 3 min
  • Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  • Transfered spnt to 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 30 min
  • Spun 8 tubes at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul 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 recombine (40ul total each)

File:2015-01-30 V6 GelQuant.jpg

  • V6: 41.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 854nM (38ul)

How I calculated concentration -->