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>Mzcai (Created page with "==CA12k_Nov2014_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering *Do double of what I normally do (96 ...") |
>Mzcai |
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(12 intermediate revisions by the same user not shown) | |||
Line 15: | Line 15: | ||
| 2X KAPA SYBG MM||50||5000 | | 2X KAPA SYBG MM||50||5000 | ||
|- | |- | ||
| | | AP1V4U (100uM)||0.4||40 | ||
|- | |- | ||
| | | AP2V4 (100uM)||0.4||40 | ||
|- | |- | ||
| H2O||49||4900 | | H2O||49||4900 | ||
Line 24: | Line 24: | ||
|} | |} | ||
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold | '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold | ||
[[File: | 800px]] | [[File:020215_CA12kNov14_ProductionPCR_V4.JPG | 800px]] | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*12 5-ml tubes (with 8 wells of PCR product each) for V4 | *12 5-ml tubes (with 8 wells of PCR product each) for V4 | ||
Line 42: | Line 42: | ||
===Qia Column Purification=== | ===Qia Column Purification=== | ||
*Purified | *Purified 12 tubes of 100ul in 12 columns following Qiagen protocol | ||
*Eluted each column with 50ul and combined all into one 1.5ml | *Eluted each column with 50ul and combined all into one 1.5ml | ||
*Measured concentration with Nanodrop: | *Measured concentration with Nanodrop: | ||
~ | ~575ul of V4 probes: 86.9 ng/ul => ~50 ug | ||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide | *Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| Amplicon|| | | Amplicon||96 | ||
|- | |- | ||
| 10X Lambda Exo Buffer||12 | | 10X Lambda Exo Buffer||12 | ||
|- | |- | ||
| Lambda Exonuclease||12 | | Lambda Exonuclease||12 | ||
|- | |- | ||
| Total|| | | Total||120 | ||
|} | |} | ||
*Incubated at 37C for 1hr | *Incubated at 37C for 1hr | ||
*Purified with | *Purified with 6 Zymo ssDNA/RNA columns | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **79.2ng/ul x 240ul = 19ug (76% yield) | ||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
====USER==== | ====USER==== | ||
*Split into 4 PCR tubes and add 5ul USER | |||
*Split into | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
Line 97: | Line 96: | ||
| 10X DpnII Buffer||2 | | 10X DpnII Buffer||2 | ||
|- | |- | ||
| 100uM RE- | | 100uM RE-DpnII_V4 guide oligo||5 | ||
|- | |- | ||
| H2O||8 | | H2O||8 | ||
Line 106: | Line 105: | ||
*Added 5ul DpnII | *Added 5ul DpnII | ||
*Incubated at 37C for overnight (~15hrs) | *Incubated at 37C for overnight (~15hrs) | ||
===Zymo Column Purification=== | ===Zymo Column Purification=== | ||
*Eluted 30ul each column ( | *Eluted 30ul each column (115ul total) | ||
*Nanodrop | *Nanodrop | ||
** | **81.2ng/ul x 115ul = 9.3ug (49% yield) | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*Run | *Run 3 gels | ||
** | **200V for 40min | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''3X Volume''' | ||
|- | |- | ||
| | | V4 Probes||115 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||115 | ||
|} | |} | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''3X Volume''' | ||
|- | |- | ||
| Low Mass Ladder|| | | Low Mass Ladder||3 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||15 | ||
|- | |- | ||
| H2O|| | | H2O||12 | ||
|- | |- | ||
| Total|| | | Total||30 | ||
|} | |} | ||
[[File:2015- | *Gel 1 | ||
[[File:2015-02-04_CA12kNov14_SizeSelect_V4_Gel1.jpg]] | |||
*Gel 2 | |||
[[File:2015-02-04_CA12kNov14_SizeSelect_V4_Gel2.jpg]] | |||
*Gel 3 | |||
[[File:2015-02-04_CA12kNov14_SizeSelect_V4_Gel3.jpg]] | |||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT. | *Put cut out gel in 0.5mL tube (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 2.0 mL tube below with pipette tip. | ||
*Added 900 ul of 1X TE buffer | *Added 900 ul of 1X TE buffer | ||
*Vortexed for | *Vortexed for 120min at 37 C in incubator | ||
*Centrifuged at 15,000 rpm for 3 min at RT | *Centrifuged at 15,000 rpm for 3 min at RT | ||
* | *Transferred the clear spnt. to 6 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 6 fresh 1.5 mL tube (~400ul per tube) | ||
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 | *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 | ||
*Vortexed and placed the 2 tubes at -80C for | *Vortexed and placed the 2 tubes at -80C for 20min | ||
*Spun | *Spun 6 tubes at 10,000rpm at 4C for 30min | ||
*Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min | *Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min | ||
*Discard supernatant and let dry in hood for 10min | *Discard supernatant and let dry in hood for 10min | ||
*Resuspend each tube with 10ul and combine | *Resuspend each tube with 10ul and combine | ||
Line 191: | Line 185: | ||
| | | | ||
|} | |} | ||
[[File:2015- | [[File:2015-02-05_CA12kNov14_V4_GelQuant.jpg|650px]] | ||
*Only use ladder bands 100bp, 200bp, | *Only use ladder bands 100bp, 200bp, to get trendline with better R^2 | ||
* | * 40ng/ul => 40ng/ul / (150nt*325Da/nt + 79Da) = 819nM (58ul) | ||
[[Media:2015- | [[Media:2015-02-05_CA12kNov14_V4_GelQuant.xlsx | How I calculated concentration]] | ||
Latest revision as of 00:15, 6 February 2015
CA12k_Nov2014_V4 Probe Production[edit]
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
- Do double of what I normally do (96 100ul PCR reactions instead of 48)
Production PCR[edit]
- 10nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon CA12k_Nov14_V7 (10nM) | 0.2 | 20 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (100uM) | 0.4 | 40 |
H2O | 49 | 4900 |
Total | 100 | 10000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:020215 CA12kNov14 ProductionPCR V4.JPG
EtOH Precipitation[edit]
- 12 5-ml tubes (with 8 wells of PCR product each) for V4
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 30min
- 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 6 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[edit]
- Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~575ul of V4 probes: 86.9 ng/ul => ~50 ug
Lambda Exonuclease Digestion[edit]
- Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 96 |
10X Lambda Exo Buffer | 12 |
Lambda Exonuclease | 12 |
Total | 120 |
- Incubated at 37C for 1hr
- Purified with 6 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 79.2ng/ul x 240ul = 19ug (76% yield)
Remove Amplification Adapters[edit]
USER[edit]
- Split into 4 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 59 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 8 |
Total | 80 |
- Incubate at 37C for 2.5 hours
DpnII[edit]
- 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[edit]
- Eluted 30ul each column (115ul total)
- Nanodrop
- 81.2ng/ul x 115ul = 9.3ug (49% yield)
PAGE Size Selection[edit]
- Run 3 gels
- 200V for 40min
Components | 3X Volume |
V4 Probes | 115 |
TBE-Urea Buffer 2X | 115 |
Components | 3X Volume |
Low Mass Ladder | 3 |
TBE-Urea Buffer 2X | 15 |
H2O | 12 |
Total | 30 |
- Gel 1
File:2015-02-04 CA12kNov14 SizeSelect V4 Gel1.jpg
- Gel 2
File:2015-02-04 CA12kNov14 SizeSelect V4 Gel2.jpg
- Gel 3
File:2015-02-04 CA12kNov14 SizeSelect V4 Gel3.jpg
EtOH Precipitation[edit]
- Put cut out gel in 0.5mL tube (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 2.0 mL tube below with pipette tip.
- Added 900 ul of 1X TE buffer
- Vortexed for 120min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 6 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 6 fresh 1.5 mL tube (~400ul per tube)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
- Vortexed and placed the 2 tubes at -80C for 20min
- Spun 6 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul 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
TBU Gel Quantification[edit]
- Dilute 2ul to 10ul with H2O
- Add 10ul 2X TBE-Urea Buffer
- Load wells with 2, 4, 6, and 8 ul
- Load Low Mass DNA ladders in 0.5, 1, and 2 ul
- Ran at 200 V for 35min
' | 0.5 ladder | 1 ladder | 2 ul | 4 ul | 6 ul | 8 ul | 2 ladder |
DNA volume | 0.5 | 1 | 0.1 | 0.2 | 0.3 | 0.4 | 2 |
H2O | 1.5 | 1 | 0.9 | 1.8 | 2.7 | 3.6 | 0 |
2X Buffer | 2 | 2 | 1 | 2 | 3 | 4 | 2 |
Total | 4 | 4 | 2 | 4 | 6 | 8 | 4 |
File:2015-02-05 CA12kNov14 V4 GelQuant.jpg
- Only use ladder bands 100bp, 200bp, to get trendline with better R^2
- 40ng/ul => 40ng/ul / (150nt*325Da/nt + 79Da) = 819nM (58ul)