Matt:LabNotes/2014-7-1: Difference between revisions
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===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide into | *Divide into four pcr tubes of 55ul with total amplicon of ~10ug | ||
{| {{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||55 | ||
|- | |- | ||
| 10X Lambda Exo Buffer||10 | | 10X Lambda Exo Buffer||10 | ||
|- | |- | ||
| Lambda Exonuclease|| | | Lambda Exonuclease||10 | ||
|- | |- | ||
| H2O|| | | H2O||25 | ||
|- | |- | ||
| | | Total||100 | ||
|} | |} | ||
*Incubated at 37C for 1hr | *Incubated at 37C for 1hr | ||
*Purified | *Purified 2 of the tubes with of Zymo ssDNA/RNA columns and 2 of the tubes with Qiagen PCR columns | ||
*Eluted with 20ul each | *Eluted with 20ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: |
Revision as of 22:52, 3 July 2014
Agi26k0gap Probe Production
Production PCR
- Make Master Mix for 46 well (6 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: |650px]]
EtOH Precipitation
- 6 5-ml tubes (with 8 wells of PCR product each) for 0gap
- 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 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:
~220ul of Agi26k0gap probes: 138.8 ng/ul => 33.3 ug
Lambda Exonuclease Digestion
- Divide into four pcr tubes of 55ul with total amplicon of ~10ug
Components | Volume |
Amplicon | 55 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 25 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified 2 of the tubes with of Zymo ssDNA/RNA columns and 2 of the tubes with Qiagen PCR columns
- Eluted with 20ul each
- Recombined and measured ssDNA with Nanodrop: