Matt:LabNotes/2015-1-15: Difference between revisions
Jump to navigation
Jump to search
>Mzcai mNo edit summary |
>Mzcai |
||
Line 87: | Line 87: | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **V4: 77.0ng/ul x 120ul = 9.24ug (% yield) | ||
** | **V7: 61.0ng/ul x 120ul = 7.32ug (% yield) | ||
<!-- | <!-- | ||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
Line 119: | Line 119: | ||
*Incubated at 94C for 2 min, then 37C for 3 min | *Incubated at 94C for 2 min, then 37C for 3 min | ||
*Added 5ul DpnII | *Added 5ul DpnII | ||
*Incubated at 37C for | *Incubated at 37C for overnight | ||
===TBU Gel: Digestion Check=== | ===TBU Gel: Digestion Check=== | ||
Line 129: | Line 129: | ||
*Digested samples (lanes 3 & 5) show significant middle bands indicating incomplete removal of amplification adapters | *Digested samples (lanes 3 & 5) show significant middle bands indicating incomplete removal of amplification adapters | ||
*To get a higher yield I continued digestion overnight | *To get a higher yield I continued digestion overnight | ||
===Zymo Column Purification=== | ===Zymo Column Purification=== |
Revision as of 01:01, 19 January 2015
CA12k_Nov2014 V4 and V7 Probe Production
- Repeat Matt:LabNotes/2015-1-5
Production PCR
- V4 master mix
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 |
- V7 master mix
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 15sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]]
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 1hr
- 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 50ul H2O
Qia Column Purification
- Purified in 6 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:
~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug ~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug
Lambda Exonuclease Digestion
- Divide each into 3 pcr tubes of 50ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 50 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 30 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 3 Zymo ssDNA/RNA columns each
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- V4: 77.0ng/ul x 120ul = 9.24ug (% yield)
- V7: 61.0ng/ul x 120ul = 7.32ug (% yield)