Matt:LabNotes/2016-11-2: Difference between revisions
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| Total||100||5000 | | 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 | '''Program''' 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold | ||
* | [[File:20161103_CA12kOct2016V4_ProductionPCR.JPG|450px]] | ||
* | *Stopped after 14 cycles | ||
[[File: | **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 | |||
[[File: | **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<!-- | |||
*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 100ul H2O | |||
===Qia Column Purification=== | |||
*8 columns elute 50ul each | |||
*Nanodrop: 287.3 ng/uL x 400uL = 114.9ug | |||
===Lambda Exo=== | |||
*Divide into 8 pcr tubes of 100ul (ideally should be <10ug each) | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| Amplicon||50 | |||
|- | |||
| 10X Lambda Exo Buffer||10 | |||
|- | |||
| Lambda Exonuclease||10 | |||
|- | |||
| H2O||30 | |||
|- | |||
| Total||100 | |||
|} | |||
*Incubated at 37C for 2hr (normally 1hr) | |||
*Purified with 8 Zymo ssDNA/RNA columns | |||
*Eluted with 40ul each | |||
*Recombined and measured ssDNA with Nanodrop: | |||
**91.1ng/ul x 320ul = 29.15ug (50.7% yield) | |||
===Remove Amplification Adapters=== | |||
====USER==== | |||
*Split into 5 PCR tubes and add 5ul USER | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| SS-amplicon||64 | |||
|- | |||
| USER||5 | |||
|- | |||
| 10X DpnII Buffer||8 | |||
|- | |||
| H2O||3 | |||
|- | |||
| Total||80 | |||
|} | |||
*Incubate at 37C for 2.5 hours | |||
====DpnII==== | |||
*Added 15ul of the following | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''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 (100ul total) | |||
*Nanodrop | |||
**147.2 ng/ul x 100ul = 14.7ug (50.4% yield) | |||
===PAGE Size Selection=== | |||
*Run 4 gels | |||
*200V for 40min | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''4X Volume''' | |||
|- | |||
| V4 Probes||95 | |||
|- | |||
| TBE-Urea Buffer 2X||95 | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''4X Volume''' | |||
|- | |||
| Low Mass Ladder||4 | |||
|- | |||
| TBE-Urea Buffer 2X||20 | |||
|- | |||
| H2O||16 | |||
|- | |||
| Total||40 | |||
|} | |||
[[File:2016-06-02_TB12kApr2016_V4_SizeSelect_Gel1.jpg|350px]]<br> | |||
[[File:2016-06-02_TB12kApr2016_V4_SizeSelect_Gel2.jpg|350px]]<br> | |||
[[File:2016-06-02_TB12kApr2016_V4_SizeSelect_Gel3.jpg|350px]]<br> | |||
[[File:2016-06-02_TB12kApr2016_V4_SizeSelect_Gel4.jpg|350px]]<br> | |||
===EtOH Precipitation=== | |||
*Put cut out gel in 8 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 (~3500ul) | |||
*Precipitated in 8 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2 | |||
*Vortexed and placed the 8 tubes at -80C for 30min | |||
*Spun 8 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=== | |||
20.2ng/ul x 74ul = 1,494.8ng | |||
20.2ng/ul / (157nt x 303.7Da/nt + 79Da) = 423nM | |||
--> |
Revision as of 03:20, 4 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 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 |
- 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