Matt:LabNotes/2015-1-15: Difference between revisions
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*Discarded supernatant and air-dried for 5 min in hood | *Discarded supernatant and air-dried for 5 min in hood | ||
*Resuspended DNA with 50ul H2O | *Resuspended DNA with 50ul H2O | ||
===Qia Column Purification=== | ===Qia Column Purification=== | ||
*Purified in 6 columns following Qiagen protocol | *Purified in 6 columns following Qiagen protocol | ||
Line 63: | Line 63: | ||
*Measured concentration with Nanodrop: | *Measured concentration with Nanodrop: | ||
~150ul of V4 probes: | ~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug | ||
~150ul of V7 probes: | ~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug | ||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide each into | *Divide each into 3 pcr tubes of 50ul with total amplicon of <10ug each | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| Amplicon|| | | Amplicon||50 | ||
|- | |- | ||
| 10X Lambda Exo Buffer||10 | | 10X Lambda Exo Buffer||10 | ||
Line 78: | Line 78: | ||
| Lambda Exonuclease||10 | | Lambda Exonuclease||10 | ||
|- | |- | ||
| H2O|| | | H2O||30 | ||
|- | |- | ||
| Total||100 | | Total||100 | ||
Line 84: | Line 84: | ||
*Incubated at 37C for 1hr | *Incubated at 37C for 1hr | ||
*Purified with | *Purified with 3 Zymo ssDNA/RNA columns each | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **ng/ul x 120ul = ug (% yield) | ||
** | **ng/ul x 120ul = ug (% yield) | ||
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===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
====USER==== | ====USER==== |
Revision as of 22:01, 18 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:
- ng/ul x 120ul = ug (% yield)
- ng/ul x 120ul = ug (% yield)