Matt:LabNotes/2016-6-20
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TB12k_Apr2016 V7 Mouse Brain Probe Preparation[edit]
Expansion PCR[edit]
Component | Volume |
Seed oligo (100X Dilution) | 15 |
F/R V7 Primer Mix (10uM) | 12 |
2X Kapa MM | 75 |
H2O | 48 |
Total | 150 |
- 50ul per PCR tube
Program
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold
File:2016-06-20 TB12kApr2016 V7 ExpansionPCR.jpg
- Purified with 3 Qiagen PCR columns (50ul PCR product each)
- Elute with 50ul H2O
- Quantify with Qubit dsDNA:
- 9.2ng/ul / (195bp*607.4Da/bp+157.9Da) = 77.57 nmol/L
- Dilute to 10nM
- 77.57nM x 144ul = 10nM x 1117ul
- Add 973ul H2O
Production PCR[edit]
- V7 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon TB12k_Apr2016_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 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold
File:2016-06-20 TB12kApr2016 V7 ProductionPCR.jpg
EtOH Precipitation[edit]
- 4 5-ml tubes (with 24 wells (3 strips) of PCR product each) for
- 1,200ul PCR product
- 3000ul 100% EtOH
- 4ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 30 min
- 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 8 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]
- 4 columns elute 50ul each
- Nanodrop: 193.0 ng/uL x 200uL = 38.6ug
Lambda Exo[edit]
- Divide into 4 pcr tubes of 100ul (ideally should be <10ug each)
Components | Volume |
Amplicon | 50 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 30 |
Total | 100 |
- Incubated at 37C for 1.5 hr (normally 1 hr)
- Purified with 4 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 68.4ng/ul x 160ul = 10.944 ug (% yield)
Remove Amplification Adapters[edit]
USER[edit]
- Split into 3 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 53 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 14 |
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_V7 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 20ul each column (60ul total)
- Nanodrop
- 111.4ng/ul x 60ul = 6.684ug (62.5% yield)
PAGE Size Selection[edit]
- Run 2 gels
- 200V for 40min
Components | 2X Volume |
V4 Probes | 60 |
TBE-Urea Buffer 2X | 60 |
Components | 2X Volume |
Low Mass Ladder | 2 |
TBE-Urea Buffer 2X | 10 |
H2O | 8 |
Total | 20 |
File:2016-06-23 TB12kApr2016V7 ProbeProduction SizeSelect.jpg
- Only saw one faint band above main band that was cut out
- That means either the USER or DpnII digestion was very complete or did not occur at all
- I am sure I used the corrent DpnII-V7 oligo
- Can run a gel comparing with 1st round amplicons to check for size next time
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 to each
- 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 450ul spnt to fresh 1.5 mL tube
- Precipitated in 4 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 8 tubes at -80C for 30min
- Spun 4 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 750ul 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[edit]
18.2ng/ul x 40ul = 728ng 18.2ng/ul / (160nt x 303.7Da/nt + 79Da) = 374 nM