Revision as of 00:52, 30 January 2015 by >Mzcai
CA12k_Nov2014 RevComp Probe Production
Expansion PCR
- 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
- V8: 48ul x 129nM = 619ul x 10nM
Gel Check
- 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
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
Production PCR
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
|
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
File:012714 CA12kNov14 ProductionPCR V6V8.JPG
EtOH Precipitation
- 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
- 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
- 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
USER
- 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
- 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
- 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
- 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
- Run 2 gels for V6 and 2 gels for V8
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
- 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)