Revision as of 22:20, 15 August 2014 by >Mzcai
Agi26k0gap Probe Production
Production PCR
- Make Master Mix for 51 well (7 strips)
- 0 gap first round amplicon -> V6 primers
Components
|
1x rxn (ul)
|
50x rxn (ul)
|
First round amplicon (10nM) |
0.2 |
10
|
2x Kapa SYBR MM |
50 |
2500
|
100uM pAP1V6U |
0.4 |
20
|
100uM AP2V6 |
0.4 |
20
|
H2O |
49 |
2450
|
Total |
100 |
5000
|
|
98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold
File:2014-08-14 Agi26k0gapProductionPCR.JPG
EtOH Precipitation
- 6 15-ml tubes (with 8.5 wells of PCR product each) for 0gap
- 850ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 85ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3000rpm at 4C for 30min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
- Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
- Eluted each column with 40ul and combined all probes into one 1.5ml
- Measured concentration with Nanodrop:
- ~225ul of Agi26k0gap probes: 317.5 ng/ul => 71.4 ug
Lambda Exonuclease Digestion
- Divide into seven pcr tubes of 32ul with total amplicon of ~10ug each
- Seventh tube had 33ul of amplicon
Components
|
Volume
|
Amplicon |
32
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
48
|
Total |
100
|
- Incubated at 37C for 1hr
- Purified with Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 89.0ng/ul x 255ul = 22.7ug (63.6% yield)
Removal of Amplification Adapters
Components
|
Volume
|
ss-amplicon |
63.5
|
USER |
5
|
H2O |
11.5
|
total |
80
|
*~5ug ssDNA per tube
- Incubated at 37C for 2 hours
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
10
|
100uM RE-DpnII guide oligo |
5
|
RE-DpnII guide oligo:
0-gap: RE DpnII V6
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for 3 hr
- Added 100ul of the following
Components
|
Volume
|
10X DpnII Buffer |
10
|
USER |
5
|
H2O |
85
|
Total |
100
|
- Incubated at 37C for overnight (~15 hrs)