Matt:LabNotes/2015-5-11: Difference between revisions
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*Incubated at 37C for 1hr | *Incubated at 37C for 1hr | ||
*Purified with 4 Zymo ssDNA/RNA columns | *Purified with 4 Zymo ssDNA/RNA columns | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **92.9 ng/ul x 160ul = 14.9 ug (67% yield) | ||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
Line 108: | Line 108: | ||
*Eluted 20ul each column (80ul total) | *Eluted 20ul each column (80ul total) | ||
*Nanodrop | *Nanodrop | ||
** | **103.7ng/ul x 80ul = 8.3ug (56% yield) | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
Line 134: | Line 134: | ||
| Total||20 | | Total||20 | ||
|} | |} | ||
[[File:2015-05-13_CA12kNov2014_V4_SizeSelect.jpg|450px]] | |||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*Put cut out gel in | *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. | *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 | *Added 450 ul of 1X TE buffer to each | ||
Line 146: | Line 144: | ||
*Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min | *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 the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | ||
*Transferred spnt to fresh 1.5 mL tube (~850ul | *Transferred spnt to fresh 1.5 mL tube (~1700ul) | ||
* | *Combined with [[Matt:LabNotes/2015-5-5#EtOH_Precipitation_2 | ~850ul from 5/5/2015]] | ||
*Precipitated in 6 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2 | |||
*Vortexed and placed the 6 tubes at -80C for 30min | |||
*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 | |||
===Qubit Quantification=== | |||
*22.5 ng/ul => 22.5 ng/ul / (150nt*325/nt + 79Da) = 461nM (60ul) |
Latest revision as of 19:22, 21 May 2015
CA12k_Nov2014_V4 Probe Production[edit]
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
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_V4 (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:20150511 CA12kNov2014 V4 ProductionPCR.JPG
EtOH Precipitation[edit]
- 8 5-ml tubes (with 12 wells of PCR product each) for V4
- 1200ul PCR product
- 3000ul 100% EtOH
- 3ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 13000rpm at 4C for 20 min (in basement ultracentrifuge)
- 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 15 min at 4C
- Discarded supernatant and air-dried for 10 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification[edit]
- Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~400ul of V4 probes: 111.1 ng/ul => ~44.4 ug
Lambda Exonuclease Digestion[edit]
- Divide into 4 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 4 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 92.9 ng/ul x 160ul = 14.9 ug (67% 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 20ul each column (80ul total)
- Nanodrop
- 103.7ng/ul x 80ul = 8.3ug (56% yield)
PAGE Size Selection[edit]
- Run 2 gels
- 200V for 40min
Components | 2X Volume |
V4 Probes | 80 |
TBE-Urea Buffer 2X | 80 |
Components | 2X Volume |
Low Mass Ladder | 2 |
TBE-Urea Buffer 2X | 10 |
H2O | 8 |
Total | 20 |
File:2015-05-13 CA12kNov2014 V4 SizeSelect.jpg
EtOH Precipitation[edit]
- 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 (~1700ul)
- Combined with ~850ul from 5/5/2015
- Precipitated in 6 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
- Vortexed and placed the 6 tubes at -80C for 30min
- 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
Qubit Quantification[edit]
- 22.5 ng/ul => 22.5 ng/ul / (150nt*325/nt + 79Da) = 461nM (60ul)