Matt:LabNotes/2015-6-3: Difference between revisions
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'''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:20150603_CA12kNov2014_V4_ProductionPCR.JPG|650px]] | |||
===QIAEX-II Purification=== | ===QIAEX-II Purification=== | ||
[http://www.qiagen.com/us/resources/download.aspx?id=13d33145-9f64-426a-a43b-394211d8cf2b&lang=en protocol] | |||
*Divide into 8 5mL tubes of 1200ul each | *Divide into 8 5mL tubes of 1200ul each | ||
*Add 3600ul (3X volume) of Buffer QX1 | *Add 3600ul (3X volume) of Buffer QX1 | ||
Line 35: | Line 37: | ||
*Wash the pellet with 500ul Buffer QX1 | *Wash the pellet with 500ul Buffer QX1 | ||
*Resuspend the pellet by vortexing | *Resuspend the pellet by vortexing | ||
*Transfer to 1.5ml tubes | |||
*Centrifuge sample for 30sec and remove supernatant again with pipette | *Centrifuge sample for 30sec and remove supernatant again with pipette | ||
*Wash the pellet twice with 500ul Buffer PE | *Wash the pellet twice with 500ul Buffer PE | ||
Line 43: | Line 46: | ||
*Repeat 20ul H2O resuspension and incubation to improve yield 10-15% | *Repeat 20ul H2O resuspension and incubation to improve yield 10-15% | ||
*Total volume: | *Total volume: 300ul | ||
*Nanodrop quantification: | *Nanodrop quantification: 155.7 ng/ul -> 46.7 ug | ||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide into 6 pcr tubes of | *Divide into 6 pcr tubes of 50ul 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||50 | ||
|- | |||
| 10X Lambda Exo Buffer||10 | |||
|- | |- | ||
| | | Lambda Exonuclease||10 | ||
|- | |- | ||
| | | H2O||30 | ||
|- | |- | ||
| Total|| | | Total||100 | ||
|} | |} | ||
*Incubated at 37C for | *Incubated at 37C for 1hr20min | ||
*Purified with 6 Zymo ssDNA/RNA columns | *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: | ||
** | **59.6ng/ul x 240ul = 14.3ug (61% yield) | ||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
Line 129: | Line 112: | ||
*Eluted 30ul each column (115ul total) | *Eluted 30ul each column (115ul total) | ||
*Nanodrop | *Nanodrop | ||
** | **51.9ng/ul x 115ul = 6.2ug (43% yield) | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*Run | *Run 2 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;"|'''2X Volume''' | ||
|- | |- | ||
| V4 Probes||115 | | V4 Probes||115 | ||
Line 145: | Line 128: | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''2X Volume''' | ||
|- | |- | ||
| Low Mass Ladder|| | | Low Mass Ladder||2 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||10 | ||
|- | |- | ||
| H2O|| | | H2O||8 | ||
|- | |- | ||
| Total|| | | Total||20 | ||
|} | |} | ||
[[File:2015-06-05_CA12kNov2014_V4_SizeSelect.jpg]] | |||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*Put cut out gel in 0.5mL | *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 | *Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. | ||
*Added | *Added 450 ul of 1X TE buffer | ||
*Vortexed for | *Vortexed for >60min 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 | *Transferred the clear spnt. to 4 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 | *Transferred spnt to 4 fresh 1.5 mL tube (~440ul per tube) | ||
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, | *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2 | ||
*Vortexed and placed the | *Vortexed and placed the 4 tubes at -80C for overnight | ||
*Spun | *Spun 4 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 10ul and combine | *Resuspend each tube with 10ul and combine | ||
=== | ===Qubit ssDNA Quantification=== | ||
21.4ng/ul => 21.4 ng/ul / (150nt*325/nt + 79Da) = 438nM (60ul) | |||
Latest revision as of 17:04, 7 June 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:20150603 CA12kNov2014 V4 ProductionPCR.JPG
QIAEX-II Purification[edit]
- Divide into 8 5mL tubes of 1200ul each
- Add 3600ul (3X volume) of Buffer QX1
- Vortex the QIAEXII for 30sec
- Add 30ul QIAEXII (30ul for every 10ug)
- Incubate at 50C while mixing for 10min
- Color should be yellow
- Centrifuge sample for 30sec
- Remove supernatant with a pipette
- Wash the pellet with 500ul Buffer QX1
- Resuspend the pellet by vortexing
- Transfer to 1.5ml tubes
- Centrifuge sample for 30sec and remove supernatant again with pipette
- Wash the pellet twice with 500ul Buffer PE
- Air-dry the pellet for 30min until the pellet turns white
- To elute, resuspend with 20ul H2O and vortexing
- Incubate at RT for 5min
- Centrifuge for 30sec and pipette out supernatant with sample
- Repeat 20ul H2O resuspension and incubation to improve yield 10-15%
- Total volume: 300ul
- Nanodrop quantification: 155.7 ng/ul -> 46.7 ug
Lambda Exonuclease Digestion[edit]
- Divide into 6 pcr tubes of 50ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 50 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 30 |
Total | 100 |
- Incubated at 37C for 1hr20min
- Purified with 6 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 59.6ng/ul x 240ul = 14.3ug (61% 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
- 51.9ng/ul x 115ul = 6.2ug (43% yield)
PAGE Size Selection[edit]
- Run 2 gels
- 200V for 40min
Components | 2X Volume |
V4 Probes | 115 |
TBE-Urea Buffer 2X | 115 |
Components | 2X Volume |
Low Mass Ladder | 2 |
TBE-Urea Buffer 2X | 10 |
H2O | 8 |
Total | 20 |
File:2015-06-05 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
- Vortexed for >60min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 4 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 4 fresh 1.5 mL tube (~440ul per tube)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
- Vortexed and placed the 4 tubes at -80C for overnight
- Spun 4 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 ssDNA Quantification[edit]
21.4ng/ul => 21.4 ng/ul / (150nt*325/nt + 79Da) = 438nM (60ul)