Matt:LabNotes/2014-8-15

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Agi26k0gap Probe Production[edit]

Production PCR[edit]

  • Make Master Mix for 51 well (7 strips)
  • 0 gap first round amplicon -> V6 primers
Components 1x rxn (ul) 50x rxn (ul)
First round amplicon (10nM) 0.2 10
2x Kapa SYBR MM 50 2500
100uM pAP1V6U 0.4 20
100uM AP2V6 0.4 20
H2O 49 2450
Total 100 5000

98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold

File:2014-08-14 Agi26k0gapProductionPCR.JPG

EtOH Precipitation[edit]

  • 6 15-ml tubes (with 8.5 wells of PCR product each) for 0gap
    • 850ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 85ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • Centrifuged at 3000rpm at 4C for 30min
  • 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 15,000rpm for 5 min on table-top centrifuge
  • 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 40ul and combined all probes into one 1.5ml
  • Measured concentration with Nanodrop:
  • ~225ul of Agi26k0gap probes: 317.5 ng/ul => 71.4 ug

Lambda Exonuclease Digestion[edit]

  • Divide into seven pcr tubes of 32ul with total amplicon of ~10ug each
    • Seventh tube had 33ul of amplicon
Components Volume
Amplicon 32
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 48
Total 100
  • Incubated at 37C for 1hr
  • Purified with Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 89.0ng/ul x 255ul = 22.7ug (63.6% yield)

Removal of Amplification Adapters[edit]

Components Volume
ss-amplicon 63.5
USER 5
H2O 11.5
total 80
 *~5ug ssDNA per tube
  • Incubated at 37C for 2 hours
  • Added 15ul of the following
Components Volume
10X DpnII Buffer 10
100uM RE-DpnII guide oligo 5
 RE-DpnII guide oligo:
 0-gap: RE DpnII V6
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for 3 hr
  • Added 100ul of the following
Components Volume
10X DpnII Buffer 10
USER 5
H2O 85
Total 100
  • Incubated at 37C for overnight (~15 hrs)

TBU Gel Check[edit]

  • Combine the 4 tubes
  • 2ul Sample + 2ul 2X Urea Loading Dye
  • 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
  • Digested sample
  • Undigested sample: 10nM 1st round amplicon

File:2014-08-17 Agi26k0gapDigestCheck.jpg

  • Brightest band below 200bp as expected

Column Purification[edit]

  • Used Zymo ssDNA/RNA columns
  • Total Eluted: 260 ul
  • Nanodrop quant: 50.8 ng/ul
  • Yield: 50.8 ng/ul x 260 ul = 13.2 ug

PAGE Size Selection[edit]

  • On 8/26/14
  • Run 4 gels of ~3.3ug ssDNA in each
    • Corresponds to 65ul in each gel
Components 1X Volume 4X Volume
Enzyme digested probes 65 260
TBE-Urea Buffer 2X 65 260
Water 0 0
Total 130 520
Components 4X Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40
  • Ran at 220V for 30min
  • Cut out brightest band below 200bp

File:2014-08-26 Agi26k0gapSizeSelectionGel1.jpg File:2014-08-26 Agi26k0gapSizeSelectionGel2.jpg File:2014-08-26 Agi26k0gapSizeSelectionGel3.jpg File:2014-08-26 Agi26k0gapSizeSelectionGel4.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 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 (~450ul per tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 8 tubes at -80C for overnight
  • 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 (70ul total)

TBU Gel Quantification[edit]

  • Diluted Agi26k0gap 10X
    • 2ul Agi26k0gap + 8ul H2O + 10ul 2X TBE-Urea buffer
  • Made DNA Low Mass Ladder with 0.5, 1, and 2 ul
Lane 1 2 3 4 5 6 7
Sample 0.5ul DNA Ladder 1ul DNA Ladder 2ul Sample 4ul Sample 6ul Sample 8ul Sample 2ul DNA Ladder

File:2014-08-27 Agi26k0gapGelQuant.jpg

  • Agi26k0gap Concentration: 82.4 ng/ul
  • Total Mass: 82.4 ng/ul * 70ul = 5.77 ug
  • Total Amount: 5.77 ug /(193nt*325 Da/nt) = 92 pmole (1.314 uM)