Matt:LabNotes/2017-5-20: Difference between revisions
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>Mzcai (Created page with "==Agi15k_Feb2017_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8 ===Notes from last time=== *Do 12 or 13 cycle...") |
>Mzcai |
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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold<br> | '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold<br> | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
Line 40: | Line 37: | ||
*Vortexed and put in -80C for overnight | *Vortexed and put in -80C for overnight | ||
*Centrifuged at | *Centrifuged at 3,000rpm at 4C for 30 min | ||
*Discarded supernatant and added 800ul of cold 80% EtOH | *Discarded supernatant and added 800ul of cold 80% EtOH | ||
*Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes | *Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes | ||
*Centrifuged 1.5ml tubes at 14,000rpm for | *Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C | ||
*Discarded supernatant and air-dried for 10 min in hood | *Discarded supernatant and air-dried for 10 min in hood | ||
*Resuspended DNA with 100ul H2O | *Resuspended DNA with 100ul H2O | ||
Line 51: | Line 48: | ||
*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: | ||
~400ul of V4 probes: | ~400ul of V4 probes: 244.2 ng/ul => ~97.64 ug | ||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
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* | *Incubate 2 tubes at 37C for 2hr and 2 tubes at 37C for 1hr | ||
*Purified with 8 Zymo ssDNA/RNA columns | *Purified with 8 Zymo ssDNA/RNA columns | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **1hr incubation: 72.3 ng/ul x 160ul = 11.6 ug | ||
*** | **2hr incubation: 81.7 ng/ul x 160ul = 13 ug | ||
*Conclusion: 2hr incubation is better | |||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
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*Eluted 20ul each column (80ul total) | *Eluted 20ul each column (80ul total) | ||
*Nanodrop | *Nanodrop | ||
** | **125.1ng/ul x 120ul = 15ug (% yield) | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
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| Total||40 | | Total||40 | ||
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===EtOH Precipitation=== | ===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. | *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 | *Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. | ||
**Had to use 2 more 1.5mL tubes to get all gel. Use 2.0mL tubes next time | |||
*Added 450 ul of 1X TE buffer to each | *Added 450 ul of 1X TE buffer to each | ||
*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 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 | *Transferred spnt to fresh 1.5 mL tube | ||
*Precipitated in | *Precipitated in 10 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2 | ||
*Vortexed and placed the | *Vortexed and placed the 10 tubes at -80C overnight | ||
*Spun | *Spun 10 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 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 | *Resuspend each tube with 8ul and combine | ||
===Qubit Quantification=== | ===Qubit Quantification=== | ||
* | *16.7 ng/ul => 16.7 ng/ul / (157nt*325/nt + 79Da) = 326.7 nM (80ul) | ||
Latest revision as of 19:50, 7 June 2017
Agi15k_Feb2017_V4 Probe Production[edit]
- Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8
Notes from last time[edit]
- Do 12 or 13 cycles Production PCR
- Experiment with 1hr vs 2hr Lambda digestion
- Think of way to maximize USER and DpnII digestion
Production PCR[edit]
- 25nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon Agi15k_Feb2017_V4 (25nM) | 0.1 | 10 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (100uM) | 0.4 | 40 |
H2O | 49 | 4910 |
Total | 100 | 10000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold
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 3,000rpm 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 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: 244.2 ng/ul => ~97.64 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 |
- Incubate 2 tubes at 37C for 2hr and 2 tubes at 37C for 1hr
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 1hr incubation: 72.3 ng/ul x 160ul = 11.6 ug
- 2hr incubation: 81.7 ng/ul x 160ul = 13 ug
- Conclusion: 2hr incubation is better
Remove Amplification Adapters[edit]
USER[edit]
- Split into 6 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[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
- 125.1ng/ul x 120ul = 15ug (% yield)
PAGE Size Selection[edit]
- Run 4 gels
- 200V for 40min
Components | 4X Volume |
V4 Probes | 120 |
TBE-Urea Buffer 2X | 120 |
Components | 4X Volume |
Low Mass Ladder | 4 |
TBE-Urea Buffer 2X | 20 |
H2O | 16 |
Total | 40 |
EtOH Precipitation[edit]
- 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.
- Had to use 2 more 1.5mL tubes to get all gel. Use 2.0mL tubes next time
- Added 450 ul of 1X TE buffer to each
- Vortexed for 120min 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 10 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
- Vortexed and placed the 10 tubes at -80C overnight
- Spun 10 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 8ul and combine
Qubit Quantification[edit]
- 16.7 ng/ul => 16.7 ng/ul / (157nt*325/nt + 79Da) = 326.7 nM (80ul)