Matt:LabNotes/2015-3-25
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CA12k_Nov2014_V4 Probe Production[edit]
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
- Do double 96 100ul PCR Reactions
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_V7 (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:032515 CA12kNov14 ProductionPCR V4.JPG
Zymo DNA Clean & Concentrator 100[edit]
- Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
- Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
- Elute with 540ul Elution buffer
- Should elute with < 400ul next time because it won't all fit in column
- Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification)
- Should elute with < 400ul next time because it won't all fit in column
- Measured concentration with Nanodrop:
~460ul of V4 probes: 71.3 ng/ul => 32.8ug
Repeat Production PCR[edit]
- 10nM 1st round amplicons
- V4 master mix made in 1 15ml centrifugal tube
- Poured into trough/reservoir and used multi-channel pipette to save time pipetting into pcr tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon CA12k_Nov14_V7 (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:032615 CA12kNov14 ProductionPCR V4.JPG
Zymo DNA Clean & Concentrator 100[edit]
- Tried using reservoir & multi-channel pipette removing product from 6 strips of pcr tubes but spilled ~1ml (product+binding buffer) pouring from reservoir into 50ml tubes
- Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
- Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
- Elute with 400ul H2O
- Somehow final volume is greater than 400ul
- Measured concentration with Nanodrop:
~410ul of V4 probes: 76.1 ng/ul => 31.2ug
Potential Reasons for Low Yield[edit]
- Second time I got 30ug when normal for EtOH precipitation method is 50ug
- Possible reasons:
- Potentially the vacuum is not strong enough to pull all solution through? (even though it's slow this is not likely)
- Losing solution on the sides of the 50ml tubes used to mix binding buffer + amplicon (definitely happening but shouldn't be 40% of amplicon)
- 400ul elution volume still too much to allow all elution buffer to get out of column membrane when centrifuged
- From Alan: Try heating up elution buffer or incubating the column with elution buffer at 37C for 5min
Lambda Exonuclease Digestion[edit]
- Combine 2 Production PCR products and Divide into 9 pcr tubes of ~110ul 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 9 Zymo ssDNA/RNA columns
- Eluted with 25ul each
- Recombined and measured ssDNA with Nanodrop:
- 100.7ng/ul x 225ul = 22.7ug (70.9% yield)
Remove Amplification Adapters[edit]
USER[edit]
- Split into 4 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 56 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 11 |
Total | 80 |
- Incubate at 37C for 2 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
- 126.8ng/ul x 115ul = 14.6ug (66% yield)
PAGE Size Selection[edit]
- Run 3 gels (4 gels ideal but ran out of 2D TBU 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:2015-03-28 CA12kNov2014 V4 SizeSelect Gel1.jpg
- Gel 2
File:2015-03-28 CA12kNov14 V4 SizeSelect Gel2.jpg
- Gel 3
File:2015-03-28 CA12kNov14 V4 SizeSelect Gel3.jpg
EtOH Precipitation[edit]
- 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 60min 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 6 tubes at -80C for overnight
- 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[edit]
- 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:2015-03-30 CA12kNov14 V4 GelQuant.jpg
- Only use ladder bands 100bp, 200bp, to get trendline with better R^2
- 70 ng/ul => 70 ng/ul / (150nt*325Da/nt + 79Da) = 1.43uM (1,433nM) (58ul)