Matt:LabNotes/2015-1-13

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CA12k_Nov2014_V7 Probe Production[edit]

Production PCR[edit]

Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V7U (100uM) 0.4 20
AP2V7 (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 File:011214 CA12kNov14 ProductionPCR V7.JPG

EtOH Precipitation[edit]

  • 6 15-ml tubes (with 8 wells of PCR product each) for V7
    • 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[edit]

  • Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~290ul of V7 probes: 93.2 ng/ul => ~27 ug

Lambda Exonuclease Digestion[edit]

  • Divide each into 3 pcr tubes of ~100ul with total amplicon of <10ug each
Components Volume
Amplicon 100
10X Lambda Exo Buffer 12.5
Lambda Exonuclease 12.5
Total 125
  • Incubated at 37C for 1hr
  • Purified with 3 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 62.9ng/ul x 120ul = 7.5ug (56% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Save 2ul for gel check later
  • Split into 2 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_V7 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)

TBU Gel: Digestion Check[edit]

  • Combine into 1 tube
  • 2ul Sample + 2ul 2X Urea Loading Dye
  • 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye

File:2015-01-14 V7 Digest GelCheck.jpg

  • Gel ran poorly for some reason but I don't have anymore undigested probe to compare to (unless I want to waste 5ul of 10nM 1st rnd amplicon) so I will just check the gel when I do size selection
    • The band to cut out should be 150bp so almost halfway between 200bp and 100bp ladder bands

Zymo Column Purification[edit]

  • Eluted 30ul each column (60ul total)
  • Nanodrop
    • 41.7ng/ul x 60ul = 2.5ug (oddly low again like last time)
      • This time 33% yield which is better than 25% yield last time

PAGE Size Selection[edit]

  • Run 1 gel for V7
    • 220V for 30min
Components 1X Volume
V7 Probes 60
TBE-Urea Buffer 2X 60
Components Volume
Low Mass Ladder 1
TBE-Urea Buffer 2X 5
H2O 4
Total 10

File:2015-01-14 SizeSelect V7.jpg

EtOH Precipitation[edit]

  • 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 2 Nanosep columns 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 2 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 2 tubes at -80C for overnight
  • Spun 2 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 combine

TBU Gel Quantification[edit]

  • Dilute 2ul to 10ul with H2O
  • Add 10ul 2X TBE-Urea Buffer
  • Load wells with 2, 4, 6, and 8 ul
  • Load Low Mass DNA ladders in 0.5, 1, and 2 ul
  • Ran at 200 V for 35min
' 0.5 ladder 1 ladder 2 ul 4 ul 6 ul 8 ul 2 ladder
DNA volume 0.5 1 0.1 0.2 0.3 0.4 2
H2O 1.5 1 0.9 1.8 2.7 3.6 0
2X Buffer 2 2 1 2 3 4 2
Total 4 4 2 4 6 8 4

File:2015-01-15 V7 GelQuant.jpg

  • Only use ladder bands 100bp, 200bp, and 400bp to get trendline with better R^2
  • 28 ng/ul => 28 ng/ul / (150nt*325Da/nt + 79Da) = 573nM (18ul)

How I calculated concentration