Revision as of 22:07, 8 January 2015 by >Mzcai
CA12k_Nov2014 Probe Production
Expansion PCR
- 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
- V7: 50ul x 142nM = 710ul x 10nM
Gel Check
- 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
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
|
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
File:010514 CA12kNov14 ProductionPCR V4V7.JPG
EtOH Precipitation
- 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
- 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
- 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
USER
- 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
- 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
- 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
- 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
- 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
- Run 2 gels for V4 and 1 gel for V7
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
|
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