Matt:LabNotes/2016-5-25

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Revision as of 23:11, 31 May 2016 by >Mzcai
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TB12k_Apr2016 Probe Preparation

  • Probe Design
  • Received 195-mer oligos 5/25/26
    • Name: TB12k_Apr2016_DARTFISH
    • Amount: 341ng
    • Physical State: Solid
  • Calculate MW of 195 nt probe = 195nt * 303.7Da/nt + 79Da = 59,300.5 g/mol
  • 341ng -> 5.75pmol

Resuspend and Aliquot

  • Resuspend in 100ul (57.5nM)
    • Pipetted 20X to mix
  • Made aliquots of 40, 40, 10, and 10 into 0.5ml Lo-Bind tubes

Serial Dilution 1000X

  • Take 1ul from a 10ul aliquot and add 9ul H2O (10X Dilution)
  • Take 5ul and add 45ul H2O (100X Dilution)
  • Take 20ul and add 180ul H2O (1000X Dilution)

Expansion PCR Test

  • NTC
Component Volume
Seed oligo 0
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 10.5
Total 25
  • Positive Control
Component Volume
CA12kNov2014_V4 1st rnd Oligos (10nM) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25
  • 1,000X Dilution
Component Volume
Seed oligo (100X Dilution) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25
  • 10,000X Dilution
Component Volume
Seed oligo (1000X Dilution) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25

Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold

File:20160526 TB12k ExpansionPCRtest.JPG

  • 1,000X final dilution was in the sweet spot for number of cycles (15-20)
  • Use that concentration and do 19 cycles

Expansion PCR

Component Volume
Seed oligo (100X Dilution) 15
F/R V4 Primer Mix (10uM) 12
2X Kapa MM 75
H2O 48
Total 150
  • 50ul per PCR tube

Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold

  • Purified with 2 Qiagen PCR columns (75ul PCR product each)
    • Elute with 50ul H2O
  • Quantify with Qubit dsDNA:
    • 10.1ng/ul / (195bp*607.4Da/bp+157.9Da) = 85.16 nmol/L
  • Dilute to 10nM
    • 85.16nM x 99ul = 10nM x 843ul
    • Add 744ul H2O

Production PCR

  • V4 master mix
Components Volume (1X) Volume (50X)
First round amplicon TB12k_Apr2016_V4 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V4U (100uM) 0.4 20
AP2V4 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold

  • Stopped after cycles

File:.JPG

EtOH Precipitation

  • 8 15-ml tubes (with 24 wells (3 strips) of PCR product each) for
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 30 min
  • 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 8 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

  • 8 columns elute 50ul each
  • Nanodrop: 287.3 ng/uL x 400uL = 114.9ug

Lambda Exo

  • Divide into 8 pcr tubes of 100ul (ideally should be <10ug each)
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 1hr
  • Purified with 8 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • ng/ul x 320ul = ug (% yield)