Matt:LabNotes/2015-3-25

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CA12k_Nov2014_V4 Probe Production[edit]

Production PCR[edit]

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
File:032515 CA12kNov14 ProductionPCR V4.JPG

Zymo DNA Clean & Concentrator 100[edit]

  • Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
    • Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
  • Elute with 540ul Elution buffer
    • Should elute with < 400ul next time because it won't all fit in column
      • Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification)
  • Measured concentration with Nanodrop:

~460ul of V4 probes: 71.3 ng/ul => 32.8ug

Repeat Production PCR[edit]

  • 10nM 1st round amplicons
  • V4 master mix made in 1 15ml centrifugal tube
  • Poured into trough/reservoir and used multi-channel pipette to save time pipetting into pcr tubes
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 File:032615 CA12kNov14 ProductionPCR V4.JPG

Zymo DNA Clean & Concentrator 100[edit]

  • Tried using reservoir & multi-channel pipette removing product from 6 strips of pcr tubes but spilled ~1ml (product+binding buffer) pouring from reservoir into 50ml tubes
  • Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
    • Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
  • Elute with 400ul H2O
    • Somehow final volume is greater than 400ul
  • Measured concentration with Nanodrop:

~410ul of V4 probes: 76.1 ng/ul => 31.2ug

Potential Reasons for Low Yield[edit]

  • Second time I got 30ug when normal for EtOH precipitation method is 50ug
  • Possible reasons:
    • Potentially the vacuum is not strong enough to pull all solution through? (even though it's slow this is not likely)
    • Losing solution on the sides of the 50ml tubes used to mix binding buffer + amplicon (definitely happening but shouldn't be 40% of amplicon)
    • 400ul elution volume still too much to allow all elution buffer to get out of column membrane when centrifuged
  • From Alan: Try heating up elution buffer or incubating the column with elution buffer at 37C for 5min

Lambda Exonuclease Digestion[edit]

  • Combine 2 Production PCR products and Divide into 9 pcr tubes of ~110ul 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 9 Zymo ssDNA/RNA columns
  • Eluted with 25ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 100.7ng/ul x 225ul = 22.7ug (70.9% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split into 4 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 56
USER 5
10X DpnII Buffer 8
H2O 11
Total 80
  • Incubate at 37C for 2 hours

DpnII[edit]

  • 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[edit]

  • Eluted 30ul each column (115ul total)
  • Nanodrop
    • 126.8ng/ul x 115ul = 14.6ug (66% yield)

PAGE Size Selection[edit]

  • Run 3 gels (4 gels ideal but ran out of 2D TBU gels)
    • 200V for 40min
Components 3X Volume
V4 Probes 115
TBE-Urea Buffer 2X 115
Components 3X Volume
Low Mass Ladder 3
TBE-Urea Buffer 2X 15
H2O 12
Total 30
  • Gel 1

File:2015-03-28 CA12kNov2014 V4 SizeSelect Gel1.jpg

  • Gel 2

File:2015-03-28 CA12kNov14 V4 SizeSelect Gel2.jpg

  • Gel 3

File:2015-03-28 CA12kNov14 V4 SizeSelect Gel3.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.
  • Transferred 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 60min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transferred the clear spnt. to 6 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 6 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 6 tubes at -80C for overnight
  • Spun 6 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 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:2015-03-30 CA12kNov14 V4 GelQuant.jpg

  • Only use ladder bands 100bp, 200bp, to get trendline with better R^2
  • 70 ng/ul => 70 ng/ul / (150nt*325Da/nt + 79Da) = 1.43uM (1,433nM) (58ul)

How I calculated concentration