Revision as of 20:09, 6 June 2016 by >Mzcai
TB12k_Apr2016 Probe Preparation[edit]
- Probe Design
- Received 195-mer oligos 5/25/26
- Name: TB12k_Apr2016_DARTFISH
- Amount: 341ng
- Physical State: Solid
- Calculate MW of 195 nt probe = 195nt * 303.7Da/nt + 79Da = 59,300.5 g/mol
- 341ng -> 5.75pmol
Resuspend and Aliquot[edit]
- Resuspend in 100ul (57.5nM)
- Made aliquots of 40, 40, 10, and 10 into 0.5ml Lo-Bind tubes
Serial Dilution 1000X[edit]
- Take 1ul from a 10ul aliquot and add 9ul H2O (10X Dilution)
- Take 5ul and add 45ul H2O (100X Dilution)
- Take 20ul and add 180ul H2O (1000X Dilution)
Expansion PCR Test[edit]
Component
|
Volume
|
Seed oligo |
0
|
F/R V4 Primer Mix (10uM) |
2
|
2X Kapa MM |
12.5
|
H2O |
10.5
|
Total |
25
|
Component
|
Volume
|
CA12kNov2014_V4 1st rnd Oligos (10nM) |
2.5
|
F/R V4 Primer Mix (10uM) |
2
|
2X Kapa MM |
12.5
|
H2O |
8
|
Total |
25
|
Component
|
Volume
|
Seed oligo (100X Dilution) |
2.5
|
F/R V4 Primer Mix (10uM) |
2
|
2X Kapa MM |
12.5
|
H2O |
8
|
Total |
25
|
Component
|
Volume
|
Seed oligo (1000X Dilution) |
2.5
|
F/R V4 Primer Mix (10uM) |
2
|
2X Kapa MM |
12.5
|
H2O |
8
|
Total |
25
|
Program
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold
File:20160526 TB12k ExpansionPCRtest.JPG
- 1,000X final dilution was in the sweet spot for number of cycles (15-20)
- Use that concentration and do 19 cycles
Expansion PCR[edit]
Component
|
Volume
|
Seed oligo (100X Dilution) |
15
|
F/R V4 Primer Mix (10uM) |
12
|
2X Kapa MM |
75
|
H2O |
48
|
Total |
150
|
Program
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold
- Purified with 2 Qiagen PCR columns (75ul PCR product each)
- Quantify with Qubit dsDNA:
- 10.1ng/ul / (195bp*607.4Da/bp+157.9Da) = 85.16 nmol/L
- Dilute to 10nM
- 85.16nM x 99ul = 10nM x 843ul
- Add 744ul H2O
Production PCR[edit]
Components
|
Volume (1X)
|
Volume (50X)
|
First round amplicon TB12k_Apr2016_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
|
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold
- Since Biorad qPCR can only do 50ul had to split into 2 96-well reactions
- First stopped after 14 cycles
File:TB12k Apr2016 V4 ProductionPCR 14cycles.JPG
- Second stopped after 12 cycles
File:TB12k Apr2016 V4 ProductionPCR 12cycles.JPG
- In the future can do 14 cycles
EtOH Precipitation[edit]
- 8 15-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]
- 8 columns elute 50ul each
- Nanodrop: 287.3 ng/uL x 400uL = 114.9ug
Lambda Exo[edit]
- Divide into 8 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 2hr (normally 1hr)
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 91.1ng/ul x 320ul = 29.15ug (50.7% yield)
Remove Amplification Adapters[edit]
- Split into 5 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
64
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
3
|
Total |
80
|
- Incubate at 37C for 2.5 hours
- 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 20ul each column (100ul total)
- Nanodrop
- 147.2 ng/ul x 100ul = 14.7ug (50.4% yield)
PAGE Size Selection[edit]
- Run 4 gels
- 200V for 40min
Components
|
4X Volume
|
V4 Probes |
95
|
TBE-Urea Buffer 2X |
95
|
Components
|
4X Volume
|
Low Mass Ladder |
4
|
TBE-Urea Buffer 2X |
20
|
H2O |
16
|
Total |
40
|
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel1.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel2.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel3.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel4.jpg
EtOH Precipitation[edit]
- Put cut out gel in 8 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 2 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 fresh 1.5 mL tube (~3500ul)
- Precipitated in 8 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 8 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]
20.2ng/ul x 74ul = 1,494.8ng
20.2ng/ul / (157nt x 303.7Da/nt + 79Da) = 423nM