Matt:LabNotes/2016-11-2: Difference between revisions
Jump to navigation
Jump to search
>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 |
||
Line 111: | Line 111: | ||
'''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
- 47.35ng/ul (3.79ug total)
- Make 12.5ul aliquots in PCR strip tubes for future
- CA12k_Oct2016 Probe Design
- May also see this probe set called TB12k_Oct2016 in my notes
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: