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

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>Mzcai
(Created page with "=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 ...")
 
>Mzcai
mNo edit summary
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'''Program'''
'''Program'''
95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold
95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold
 
[[File:20161102_CA12kOct2016V4_ExpansionPCRtest.JPG|450px]]


==Expansion PCR V4==
==Expansion PCR V4==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''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 24 -> 72C 2min -> 15C hold
*Purified with 2 Qiagen PCR columns (50ul 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-->

Revision as of 23:39, 2 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 24 -> 72C 2min -> 15C hold

  • Purified with 2 Qiagen PCR columns (50ul PCR product each)
    • Elute with 50ul H2O
  • Quantify with Qubit dsDNA: