Revision as of 21:47, 2 June 2017 by >Mzcai
Agi15k_Feb2017_V4 Probe Production
- Do 12 or 13 cycles Production PCR
- Experiment with 1hr vs 2hr Lambda digestion
- Think of way to maximize USER and DpnII digestion
Production PCR
Components
|
Volume (1X)
|
Volume (100X)
|
First round amplicon Agi15k_Feb2017_V4 (25nM) |
0.1 |
10
|
2X KAPA SYBG MM |
50 |
5000
|
AP1V4U (100uM) |
0.4 |
40
|
AP2V4 (100uM) |
0.4 |
40
|
H2O |
49 |
4910
|
Total |
100 |
10000
|
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold
EtOH Precipitation
- 8 5-ml tubes (with 12 wells of PCR product each) for V4
- 1200ul PCR product
- 3000ul 100% EtOH
- 3ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3,000rpm 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 10 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~400ul of V4 probes: 244.2 ng/ul => ~97.64 ug
Lambda Exonuclease Digestion
- Divide into 4 pcr tubes of ~100ul with total amplicon of <10ug each
Components
|
Volume
|
Amplicon |
96
|
10X Lambda Exo Buffer |
12
|
Lambda Exonuclease |
12
|
Total |
120
|
- Incubate 2 tubes at 37C for 2hr and 2 tubes at 37C for 1hr
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 1hr incubation: 72.3 ng/ul x 160ul = 11.6 ug
- 2hr incubation: 81.7 ng/ul x 160ul = 13 ug
- Conclusion: 2hr incubation is better
Remove Amplification Adapters
USER
- Split into 6 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
52
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
15
|
Total |
80
|
- Incubate at 37C for 2.5 hours
DpnII
- 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
- Eluted 20ul each column (80ul total)
- Nanodrop
- 125.1ng/ul x 120ul = 15ug (% yield)
PAGE Size Selection
Components
|
4X Volume
|
V4 Probes |
120
|
TBE-Urea Buffer 2X |
120
|
Components
|
4X Volume
|
Low Mass Ladder |
4
|
TBE-Urea Buffer 2X |
20
|
H2O |
16
|
Total |
40
|
EtOH Precipitation
- 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.
- Had to use 2 more 1.5mL tubes to get all gel. Use 2.0mL tubes next time
- Added 450 ul of 1X TE buffer to each
- Vortexed for 120min 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
- Precipitated in 10 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
- Vortexed and placed the 10 tubes at -80C overnight
- Spun 10 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul 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 8ul and combine
Qubit Quantification
- 16.7 ng/ul => 16.7 ng/ul / (157nt*325/nt + 79Da) = 326.7 nM (80ul)