Matt:LabNotes/2015-1-26
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CA12k_Nov2014 RevComp Probe Production[edit]
- Production of Padlock Probes targeting cDNA (V6 and V8 primers) of Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
Expansion PCR[edit]
- Matt:LabNotes/2014-12-18 conclusion: 21 cycles of PCR starting with 100nM seed oligos
- Make 2 tubes:
- V6 primers - CA12k_Nov2014_Contig_RevComp
- V8 primers - CA12k_Nov2014_Const_RevComp
' | Contig of Exons RevComp(V6) | Constitutive Exons RevComp(V8) |
CA12k_Nov2014 Seed Oligos (900nM) | 15 | 15 |
2X KAPA SYBG MM | 67.5 | 67.5 |
100uM AP1V6U | 0.6 | 0 |
100uM AP2V6 | 0.6 | 0 |
100uM AP1V8U | 0 | 0.6 |
100uM AP2V8 | 0 | 0.6 |
H2O | 51.3 | 51.3 |
Total | 135 | 135 |
Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold
File:012614 CA12kNov14 ExpansionPCR V6V8.JPG
- Did not think V6 amplified correctly (possibly due to underpipetting a primer) but V4 showed a similar curve
- Nanodrop also shows V6 amplified as much or more than V4
- Purified each with 1 Qiagen column and eluted with 50ul H2O
- Nanodrop:
- V6: 11.8 ng/ul -> 11.8 ng/ul / (188bp*660Da/bp) = 95nM
- V8: 16.2 ng/ul -> 16.2 ng/ul / (190bp*660Da/bp) = 129nM
- Diluted to 10nM
- V6: 48ul x 95nM = 456ul x 10nM
- Add 408ul H2O
- V8: 48ul x 129nM = 619ul x 10nM
- Add 571ul H2O
- V6: 48ul x 95nM = 456ul x 10nM
Gel Check[edit]
- Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye
- Use 2ul 10nM Agi26k0gap expansion PCR'd probes as positive control
File:2015-01-26 1stRndAmplicon V6V8 Gel.jpg
- Apparently Nanodrop is not accurate enough at low conc
- V6 is missing a band at 188bp...
- Try Expansion PCR with V6 primers on the last of the seed oligos
V6 Expansion PCR Try 2[edit]
Components | Volume |
CA12k_Nov2014 Seed Oligos (900nM) | 14 |
2X KAPA SYBG MM | 50 |
100uM AP1V6U | 1 |
100uM AP2V6 | 1 |
H2O | 34 |
Total | 100 |
Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold File:012614 CA12kNov14 ExpansionPCR V6 try2.JPG
- Stopped after 22 cycles
- Purified each with 1 Qiagen column and eluted with 50ul H2O
- Nanodrop:
- V6: 17.9 ng/ul -> 17.9 ng/ul / (188bp*660Da/bp) = 144nM
- Diluted to 10nM
- V6: 48ul x 144nM = 691ul x 10nM
- Add 643ul H2O
- V6: 48ul x 144nM = 691ul x 10nM
Production PCR[edit]
- V6 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V6 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V6U (100uM) | 0.4 | 20 |
AP2V6 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
- V8 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V8 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V8U (100uM) | 0.4 | 20 |
AP2V8 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold
- Stopped after 15 cycles
File:012714 CA12kNov14 ProductionPCR V6V8.JPG
EtOH Precipitation[edit]
- 6 5-ml tubes (with 8 wells of PCR product each) for V6 and 6 tubes for V8
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 1hr
- Centrifuged at 3000rpm at 4C for 30 min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification[edit]
- Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
- Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
- Measured concentration with Nanodrop:
~290ul of V6 probes: 158.9 ng/ul => ~ 46 ug
~290ul of V8 probes: 183.1 ng/ul => ~ 53 ug
Lambda Exonuclease Digestion[edit]
- Divide each into 6 pcr tubes of 48ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 48 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 32 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 6 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- V6: 64.0ng/ul x 240ul = 15.4ug (67% yield)
- V8: 65.0ng/ul x 240ul = 15.6ug (59% yield)
Remove Amplification Adapters[edit]
USER[edit]
- Split each set into 3 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 80 |
USER | 5 |
Total | 85 |
- Incubate at 37C for 3 hours
DpnII[edit]
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 10 |
100uM RE-DpnII guide oligo | 5 |
RE-DpnII guide oligo: V6: RE DpnII V6 V8: RE DpnII V8
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (16 hrs)
TBU Gel: Digestion Check[edit]
- Combine into 1 tube for each set
- 2ul Digested Sample + 2ul 2X Urea Loading Dye
- 5ul Undigested sample (10nM V8 1st round amplicon) + 5ul 2X Urea Loading Dye
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
File:2015-01-28 V6V8 Digest GelCheck.jpg
Zymo Column Purification[edit]
- 3 columns for each set
- Eluted 20ul each column (60ul total)
- Nanodrop
- V6: 137.3ng/ul x 60ul = 8.2ug
- V8: 116.2ng/ul x 60ul = 6.9ug
PAGE Size Selection[edit]
- Run 2 gels for V6 and 2 gels for V8
- 200V for 35min
Components | 2X Volume |
V6 Probes | 60 |
H2O | 60 |
TBE-Urea Buffer 2X | 120 |
Components | 2X Volume |
V8 Probes | 60 |
H2O | 60 |
TBE-Urea Buffer 2X | 120 |
Components | Volume |
Low Mass Ladder | 4 |
TBE-Urea Buffer 2X | 20 |
H2O | 16 |
Total | 40 |
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.
- Transfered 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 60 min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transfered the clear spnt. to 8 Nanosep column and centrifuged at 15,000 rpm for 3 min
- Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transfered spnt to 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 8 tubes at -80C for 30 min
- Spun 8 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul 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 recombine (40ul total each)
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 |
- V6
File:2015-01-30 V6 GelQuant.jpg
- V8
File:2015-01-30 V8 GelQuant.jpg
- Only use ladder bands 100bp, 200bp, and 400bp because others were too saturated and non-linear
- V6: 41.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 854nM (38ul)
- V8: 42.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 874nM (38ul)