Revision as of 19:39, 1 August 2016 by >Mzcai
TB12k_Apr2016 V4 Probe Preparation[edit]
Production PCR[edit]
Components
|
Volume (1X)
|
Volume (50X)
|
First round amplicon TB12k_Apr2016_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
|
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold
- No curve because 100ul reaction volume only works in Biorad PCR machines
EtOH Precipitation[edit]
- 8 15-ml tubes (with 24 wells (3 strips) of PCR product each) for
- 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 8 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[edit]
- 8 columns elute 50ul each
- Nanodrop: 313.6 ng/uL x 400uL = 125.44ug
Lambda Exo[edit]
- Divide into 8 pcr tubes of 100ul (ideally should be <10ug each)
Components
|
Volume
|
Amplicon |
50
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
30
|
Total |
100
|
- Incubated at 37C for 2hr (normally 1hr)
- Purified with 8 Zymo ssDNA/RNA columns
- Dropped one column so only 7
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 118.7ng/ul x 280ul = 33.24ug
Remove Amplification Adapters[edit]
- Split into 5 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
56
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
11
|
Total |
80
|
- Incubate at 37C for 2.5 hours
- 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[edit]
- Eluted 20ul each column (100ul total)
- Nanodrop
- 193.7 ng/ul x 100ul = 19.37 ug (% yield)