Revision as of 03:06, 10 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 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 50ul H2O (usually 100ul but because only did 5ml instead of 10ml production PCR this time)
Qia Column Purification
- 4 columns elute 50ul each
- Nanodrop: 208.8 ng/uL x 200uL = 41.76ug
Lambda Exo
- Divide into 4 pcr tubes of 100ul (ideally should be <10ug each)
Components
|
Volume
|
Amplicon |
50
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
30
|
Total |
100
|
- Incubated at 37C for 1hr
- Purified with 4 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 96.8ng/ul x 160ul = 15.49ug (74% yield)
Remove Amplification Adapters
- Save 4ul for gel check of size
USER
- Split into 3 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
52
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
15
|
Total |
80
|
- Incubate at 37C for 2.5 hours
DpnII
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
2
|
100uM RE-DpnII_V4 guide oligo |
5
|
H2O |
8
|
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (~15hrs)
Zymo Column Purification
- Eluted 20ul each column (60ul total)
- Nanodrop
- 87.3 ng/ul x 60ul = 5.2ug (34% yield lower than normal)
Gel Check
- Pre-run 12 well TBU gel for 20min at 250V
- Rinse wells
- Ladder
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X TBU Loading Dye
- User/DpnII-Digested
- 2ul Sample + 2ul 2X TBU Loading Dye
- Lambda-Digested
- 2ul Sample + 2ul 2X TBU Loading Dye
- Pre-heat samples to 75C for 10min and then put on ice
- Load samples in middle lanes of gel
- Run for 33min at 210V
File:2016-11-08 CA12kOct2016 V4 GelCheck.jpg
PAGE Size Selection
- Run 2 gels
- 200V for 40min
Components
|
2X Volume
|
V4 Probes |
60
|
TBE-Urea Buffer 2X |
60
|
Components
|
2X Volume
|
Low Mass Ladder |
2
|
TBE-Urea Buffer 2X |
10
|
H2O |
8
|
Total |
20
|
[[File:|350px]]
[[File:|350px]]
EtOH Precipitation
- Put cut out gel in 4 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
- Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
- Added 450 ul of 1X TE buffer to each
- Vortexed for 60min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
- Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transferred spnt to fresh 1.5 mL tube
- Precipitated in 4 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 4 tubes at -80C for 30min
- Spun 4 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 750ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
- Discard supernatant and let dry in hood for 10min
- Resuspend each tube with 10ul and combine
Qubit ssDNA
990pg/ul x 40ul = 39.6ng
990pg/ul / (157 x 303.7Da/nt + 79Da) = 20.7nM