Matt:LabNotes/2016-11-2: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
mNo edit summary
>Mzcai
Line 175: Line 175:
**120ul 3M NaOAc pH 5.2-5.5
**120ul 3M NaOAc pH 5.2-5.5


*Vortexed and put in -80C for 30 min<!--
*Vortexed and put in -80C for overnight
*Centrifuged at 3000rpm at 4C for 30 min
*Centrifuged at 3000rpm at 4C for 30 min
*Discarded supernatant and added 800ul of cold 80% EtOH
*Discarded supernatant and added 800ul of cold 80% EtOH
Line 182: Line 182:
*Discarded supernatant and air-dried for 5 min in hood
*Discarded supernatant and air-dried for 5 min in hood
*Resuspended DNA with 100ul H2O
*Resuspended DNA with 100ul H2O
 
<!--
===Qia Column Purification===
===Qia Column Purification===
*8 columns elute 50ul each
*8 columns elute 50ul each

Revision as of 18:44, 7 November 2016

CA12k_Oct2016 Probe Preparation

  • Received CA12k_Oct2016_Oligos2Order_FINAL in 80ul TE Buffer
    • 47.35ng/ul (3.79ug total)
      • Calculate MW of 195 nt probe = 195nt * 303.7D/nt + 79D = 59,300.5 g/mol
    • 47.35 ng/ul * nmol/59300.5ng = 0.0007985 nmol/ul = 0.8 pmol/ul = 0.8 uM
  • Make 12.5ul aliquots in PCR strip tubes for future
Probeset Amplification # Probes # Barcodes # Genes
Human Brain V4 4,978 391 368
Mouse Brain V7 3,602 342 319
Mouse Embryo V6 2,469 251 220
Lung Cancer V8 105 105 85

Expansion PCR Test V4

  • NTC
Component Volume
Seed oligo 0
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 10.5
Total 25
  • Pos Control
Component Volume
TB12k_Apr2016 Seed oligo (100X Dilution) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25
  • 50nM CA12k_Oct2016
Component Volume
Seed oligo (800nM) 1.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 9
Total 25
  • 100nM CA12k_Oct2016
Component Volume
Seed oligo (800nM) 3.125
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 7.375
Total 25

Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold File:20161102 CA12kOct2016V4 ExpansionPCRtest.JPG

Expansion PCR V4

Component Volume
Seed oligo (800nM) 12.5
F/R V4 Primer Mix (10uM) 8
2X Kapa MM 50
H2O 29.5
Total 100
  • 50ul per PCR tube

Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 23 -> 72C 2min -> 15C hold File:20161102 CA12kOct2016V4 ExpansionPCR.JPG

  • Purified with 2 Qiagen PCR columns (50ul PCR product each)
    • Elute with 50ul H2O
  • Quantify with Qubit dsDNA:
    • 2.6ng/ul / (195bp*607.4Da/bp+157.9Da) = 21.9 nmol/L
  • Dilute to 10nM
    • 21.9nM x 99ul = 10nM x 216.8ul
    • Add 117.8ul H2O

Production PCR

  • V4 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Oct2016_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
  • Did one plate - 96 wells 50ul each

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold File:20161103 CA12kOct2016V4 ProductionPCR.JPG

  • Stopped after 14 cycles
    • Based on touchscreen image curve probably peaked at cycle 13 and started to go down just a little at cycle 14
  • Around cycle 12 the PCR curve started to look tilted like in the image here
    • It started with about half the wells with no obvious pattern which wells
    • Right at cycle 14 all the remaining normal curves also became tilted

EtOH Precipitation

  • 8 5-ml tubes each with
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 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 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