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

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
mNo edit summary
Line 139: Line 139:
**21.9nM x 99ul = 10nM x 216.8ul
**21.9nM x 99ul = 10nM x 216.8ul
**Add 117.8ul H2O
**Add 117.8ul H2O
==Production PCR==
*V4 master mix
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume (1X)'''
| align="center" style="background:#f0f0f0;"|'''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
|}
'''Program''' 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold
*Since Biorad qPCR can only do 50ul had to split into 2 96-well reactions
*First stopped after 14 cycles
[[File:TB12k_Apr2016_V4_ProductionPCR_14cycles.JPG | 650px]]
*Second stopped after 12 cycles
[[File:TB12k_Apr2016_V4_ProductionPCR_12cycles.JPG | 650px]]
*'''In the future can do 14 cycles'''

Revision as of 21:10, 3 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

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

  • Since Biorad qPCR can only do 50ul had to split into 2 96-well reactions
  • First stopped after 14 cycles

File:TB12k Apr2016 V4 ProductionPCR 14cycles.JPG

  • Second stopped after 12 cycles

File:TB12k Apr2016 V4 ProductionPCR 12cycles.JPG

  • In the future can do 14 cycles