Matt:LabNotes/2015-6-3: Difference between revisions
Jump to navigation
Jump to search
>Mzcai m (→DpnII) |
>Mzcai |
||
Line 112: | Line 112: | ||
*Eluted 30ul each column (115ul total) | *Eluted 30ul each column (115ul total) | ||
*Nanodrop | *Nanodrop | ||
**51.9ng/ul x 115ul = 6.2ug ( | **51.9ng/ul x 115ul = 6.2ug (43% yield) | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== |
Revision as of 19:42, 5 June 2015
CA12k_Nov2014_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
Production PCR
- 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
- 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
- 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
USER
- 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
- 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
- Eluted 30ul each column (115ul total)
- Nanodrop
- 51.9ng/ul x 115ul = 6.2ug (43% yield)
PAGE Size Selection
- 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
- 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
- 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)