Matt:LabNotes/2017-4-12: Difference between revisions
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==Agi15k_Feb2017_V4 Probe Production== | ==Agi15k_Feb2017_V4 Probe Production== | ||
*Production of Padlock Probes (V4) [[Matt:LabNotes/2017-2-8]] | *Production of Padlock Probes (V4) [[Matt:LabNotes/2017-2-8]] | ||
===Notes for next time=== | |||
*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=== | ===Production PCR=== | ||
Line 22: | Line 27: | ||
| Total||100||10000 | | Total||100||10000 | ||
|} | |} | ||
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold | '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold<br> | ||
[[File:20170412_Agi15kFeb2017_ProductionPCR.png | 600px]] | [[File:20170412_Agi15kFeb2017_ProductionPCR.png | 600px]] | ||
*Do 12 or 13 cycles next time | *Do 12 or 13 cycles next time | ||
Line 45: | Line 50: | ||
*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: 327.7 ng/ul => ~131 ug | |||
~400ul of V4 probes: | |||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
Line 63: | Line 67: | ||
|} | |} | ||
*Incubated at 37C for | *Incubated at 37C for 2hr | ||
*Purified with | *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: | ||
** | **95.1 ng/ul x 320ul = 30.4 ug (46.5% yield) | ||
***Maybe try less lambda digestion time? or more? | |||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
====USER==== | ====USER==== | ||
*Split into | *Split into 6 PCR tubes and add 5ul USER | ||
{| {{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''' | ||
|- | |- | ||
| SS-amplicon|| | | SS-amplicon||52 | ||
|- | |- | ||
| USER||5 | | USER||5 | ||
Line 82: | Line 87: | ||
| 10X DpnII Buffer||8 | | 10X DpnII Buffer||8 | ||
|- | |- | ||
| H2O|| | | H2O||15 | ||
|- | |- | ||
| Total||80 | | Total||80 | ||
Line 109: | Line 114: | ||
*Eluted 20ul each column (80ul total) | *Eluted 20ul each column (80ul total) | ||
*Nanodrop | *Nanodrop | ||
** | **114.9ng/ul x 120ul = 13.8ug (% yield) | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*Run | *Run 4 gels | ||
**200V for 40min | **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;"|'''4X Volume''' | ||
|- | |- | ||
| V4 Probes|| | | V4 Probes||120 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||120 | ||
|} | |} | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''4X Volume''' | ||
|- | |- | ||
| Low Mass Ladder|| | | Low Mass Ladder||4 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||20 | ||
|- | |- | ||
| H2O|| | | H2O||16 | ||
|- | |- | ||
| Total|| | | Total||40 | ||
|} | |} | ||
*Did not save images but could see digestion was incomplete (<1% completely undigested, ~50% half digested) | |||
**Increase USER incubation? Increase DpnII guide oligo concentration? | |||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*Put cut out gel in | *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 2 mL tube below with pipette tip. | ||
*Added 450 ul of 1X TE buffer to each | *Added 450 ul of 1X TE buffer to each | ||
*Vortexed for 60min at 37 C in incubator | *Vortexed for 60min at 37 C in incubator | ||
Line 145: | Line 151: | ||
*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 8 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2 | |||
*Precipitated in | *Vortexed and placed the 6 tubes at -80C overnight | ||
*Vortexed and placed the 6 tubes at -80C | *Spun 8 tubes at 10,000rpm at 4C for 30min | ||
*Spun | |||
*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 | ||
Line 155: | Line 160: | ||
===Qubit Quantification=== | ===Qubit Quantification=== | ||
* | *10.3 ng/ul => 10.3 ng/ul / (157nt*325/nt + 79Da) = 201.5 nM (80ul) | ||
Latest revision as of 21:53, 18 April 2017
Agi15k_Feb2017_V4 Probe Production[edit]
- Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8
Notes for next 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 14 -> 72C 2min -> 15C hold
File:20170412 Agi15kFeb2017 ProductionPCR.png
- Do 12 or 13 cycles next time
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 10,000rpm 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 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: 327.7 ng/ul => ~131 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 2hr
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 95.1 ng/ul x 320ul = 30.4 ug (46.5% yield)
- Maybe try less lambda digestion time? or more?
- 95.1 ng/ul x 320ul = 30.4 ug (46.5% yield)
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
- 114.9ng/ul x 120ul = 13.8ug (% 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 |
- Did not save images but could see digestion was incomplete (<1% completely undigested, ~50% half digested)
- Increase USER incubation? Increase DpnII guide oligo concentration?
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 2 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 8 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 overnight
- Spun 8 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]
- 10.3 ng/ul => 10.3 ng/ul / (157nt*325/nt + 79Da) = 201.5 nM (80ul)