Revision as of 03:20, 4 November 2016 by >Mzcai
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
Component
|
Volume
|
Seed oligo |
0
|
F/R V4 Primer Mix (10uM) |
2
|
2X Kapa MM |
12.5
|
H2O |
10.5
|
Total |
25
|
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
|
Component
|
Volume
|
Seed oligo (800nM) |
1.5
|
F/R V4 Primer Mix (10uM) |
2
|
2X Kapa MM |
12.5
|
H2O |
9
|
Total |
25
|
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
|
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)
- 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
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 30 min