Revision as of 01:33, 14 November 2016 by >Mzcai
CA12k_Oct2016 V4 Probe Preparation
Production PCR
Components
|
Volume (1X)
|
Volume (50X)
|
First round amplicon CA12k_Oct2016_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
|
- Did one plate - 96 wells 100ul each
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold
450px
EtOH Precipitation
- 16 5-ml tubes each with
- 1,200ul PCR product
- 3000ul 100% EtOH
- 4ul GlycoBlue
- 120ul 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 16 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
- 16 columns elute 50ul each
- Nanodrop: 170.3 ng/uL x 800uL = 136.24ug
Lambda Exo
- Divide into 14 pcr tubes of 100ul (ideally should be <10ug each)
Components
|
Volume
|
Amplicon |
57
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
23
|
Total |
100
|
- Incubated at 37C for 1hr
- Purified with 14 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 72.4ng/ul x 560ul = 40.54ug (60% yield)
Remove Amplification Adapters
USER
- Split into 9 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
62.2
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
4.8
|
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)