Matt:LabNotes/2015-1-5
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CA12k_Nov2014 Probe Production[edit]
- Production of Padlock Probes targeting cDNA (V4 and V7 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:
- V4 primers - CA12k_Nov2014_Contig
- V7 primers - CA12k_Nov2014_Const
' | Contig of Exons (V4) | Constitutive Exons (V7) |
CA12k_Nov2014 Seed Oligos (900nM) | 15 | 15 |
2X KAPA SYBG MM | 67.5 | 67.5 |
100uM AP1V4U | 0.6 | 0 |
100uM AP2V4 | 0.6 | 0 |
100uM AP1V7U | 0 | 0.6 |
100uM AP2V7 | 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:010514 CA12kNov14 ExpansionPCR V4V7.JPG
- Purified each with 1 Qiagen column and eluted with 50ul H2O
- Nanodrop:
- V4: 10.8 ng/ul -> 10.8 ng/ul / (188bp*660Da/bp) = 87nM
- V7: 17.8 ng/ul -> 17.8 ng/ul / (190bp*660Da/bp) = 142nM
- Diluted to 10nM
- V4: 50ul x 87nM = 435ul x 10nM
- Add 385ul H2O
- V7: 50ul x 142nM = 710ul x 10nM
- Add 660ul H2O
- V4: 50ul x 87nM = 435ul x 10nM
Gel Check[edit]
- Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye
- Use 10nM Agi26k0gap expansion PCR'd probes as positive control
- Although band is very faint it can be seen at slightly under 200bp
File:2015-01-06 1stRndAmplicon V4V7 Gel.jpg
Production PCR[edit]
- V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V4 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V4U (100uM) | 0.4 | 20 |
AP2V4 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
- V7 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V7 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V7U (100uM) | 0.4 | 20 |
AP2V7 (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:010514 CA12kNov14 ProductionPCR V4V7.JPG
EtOH Precipitation[edit]
- 6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- 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 V4 probes into one 1.5ml and all V7 probes into another
- Measured concentration with Nanodrop:
~290ul of V4 probes: 127.8 ng/ul => ~37 ug ~290ul of V7 probes: 113.9 ng/ul => ~33 ug
Lambda Exonuclease Digestion[edit]
- Divide each into 4 pcr tubes of 72.5ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 72.5 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 7.5 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 4 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 66.5ng/ul x 160ul = 10.6ug (57.3% yield)
- 56.9ng/ul x 160ul = 9.1ug (55.2% yield)
Remove Amplification Adapters[edit]
USER[edit]
- Split each set into 2 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 80 |
USER | 5 |
Total | 85 |
- Incubate at 37C for 2.5 hours
DpnII[edit]
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 10 |
100uM RE-DpnII guide oligo | 5 |
RE-DpnII guide oligo: V4: RE DpnII V4 V7: RE DpnII V7
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for 4 hr
TBU Gel: Digestion Check[edit]
- Combine into 1 tube for each set
- 2ul Digested Sample + 2ul 2X Urea Loading Dye
- 5ul Undigested sample (10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
File:2015-01-07 V4V7 Digest GelCheck.jpg
- Digested samples (lanes 3 & 5) show significant middle bands indicating incomplete removal of amplification adapters
- To get a higher yield I continued digestion overnight
Continue Digestion[edit]
- Add 100ul of the following to each tube
Components | Volume |
10X DpnII Buffer | 10 |
USER | 5 |
H2O | 85 |
Total | 100 |
- Incubated at 37C for overnight (15 hrs)
Zymo Column Purification[edit]
- 4 columns for each set
- Eluted 30ul each column (120ul total)
- Nanodrop
- V4: 45.0ng/ul x 120ul = 5.4ug
- V7: 22.8ng/ul x 120ul = 2.7ug (oddly low but checked with nanodrop twice)
PAGE Size Selection[edit]
- Run 2 gels for V4 and 1 gel for V7
- 220V for 30min
Components | 2X Volume |
V4 Probes | 120 |
TBE-Urea Buffer 2X | 120 |
Components | 1X Volume |
V7 Probes | 120 |
TBE-Urea Buffer 2X | 120 |
Components | Volume |
Low Mass Ladder | 3 |
TBE-Urea Buffer 2X | 15 |
H2O | 12 |
Total | 30 |
- V4 Gel 1
File:2015-01-08 SizeSelect V4 Gel1.jpg
- V4 Gel 2
File:2015-01-08 SizeSelect V4 Gel2.jpg
- V7 Gel 1
- Had trouble seeing blade and may have cut some extra above where I should have. However a mitigating factor is that V7 was digested much more completely than V4 based on intensity of band before cutting
File:2015-01-08 SizeSelect V7 Gel1.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.
- 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 6 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 6 tubes at -80C for overnight
- Spun 6 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 for V4 and 20ul for V7)
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:2015-01-09 V4 GelQuant.jpg File:2015-01-09 V7 GelQuant.jpg
- Only use ladder bands 100bp, 200bp, and 400bp because others were too saturated and non-linear
- V4: 10.7 ng/ul => 10.7 ng/ul / (150nt*325Da/nt + 79Da) = 219nM (38ul)
- V7: 6.2 ng/ul => 6.2 ng/ul / (150nt*325Da/nt + 79Da) = 127nM (18ul)