Matt:LabNotes/2015-1-13: Difference between revisions
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>Mzcai mNo edit summary |
>Mzcai mNo edit summary |
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**80ul 3M NaOAc pH 5.2-5.5 | **80ul 3M NaOAc pH 5.2-5.5 | ||
*Vortexed and put in -80C for overnight | *Vortexed and put in -80C for overnight | ||
*Centrifuged at 3000rpm at 4C for 30 min | *Centrifuged at 3000rpm at 4C for 30 min | ||
*Discarded supernatant and added 800ul of cold 80% EtOH | *Discarded supernatant and added 800ul of cold 80% EtOH | ||
*Transferred DNA pellet (with 1ml pipette tip) to | *Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes | ||
*Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C | *Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C | ||
*Discarded supernatant and air-dried for 5 min in hood | *Discarded supernatant and air-dried for 5 min in hood | ||
Line 41: | Line 41: | ||
===Qia Column Purification=== | ===Qia Column Purification=== | ||
*Purified | *Purified 6 tubes of 100ul in 6 columns following Qiagen protocol | ||
*Eluted each column with 50ul and combined all | *Eluted each column with 50ul and combined all into one 1.5ml | ||
*Measured concentration with Nanodrop: | *Measured concentration with Nanodrop: | ||
~290ul of V7 probes: 93.2 ng/ul => ~27 ug | |||
~290ul of V7 probes: | |||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide each into | *Divide each into 3 pcr tubes of ~100ul 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||100 | ||
|- | |- | ||
| 10X Lambda Exo Buffer|| | | 10X Lambda Exo Buffer||12.5 | ||
|- | |- | ||
| Lambda Exonuclease|| | | Lambda Exonuclease||12.5 | ||
|- | |- | ||
| Total||125 | |||
| Total|| | |||
|} | |} | ||
*Incubated at 37C for 1hr | *Incubated at 37C for 1hr | ||
*Purified with | *Purified with 3 Zymo ssDNA/RNA columns | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **62.9ng/ul x 120ul = 7.5ug (56% yield) | ||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
====USER==== | ====USER==== | ||
*Split | *Save 2ul for gel check later | ||
*Split into 2 PCR tubes and add 5ul USER | |||
{| {{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''' | ||
|- | |- | ||
| SS-amplicon|| | | SS-amplicon||59 | ||
|- | |- | ||
| USER||5 | | USER||5 | ||
|- | |- | ||
| Total|| | | 10X DpnII Buffer||8 | ||
|- | |||
| H2O||8 | |||
|- | |||
| Total||80 | |||
|} | |} | ||
*Incubate at 37C for | *Incubate at 37C for 1.5 hours | ||
====DpnII==== | ====DpnII==== | ||
*Added 15ul of the following | *Added 15ul of the following | ||
Line 92: | Line 93: | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| 10X DpnII Buffer|| | | 10X DpnII Buffer||2 | ||
|- | |||
| 100uM RE-DpnII_V7 guide oligo||5 | |||
|- | |||
| H2O||8 | |||
|- | |- | ||
|} | |} | ||
*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=== | ||
*Combine into 1 tube for each set | *Combine into 1 tube for each set |
Revision as of 01:31, 14 January 2015
CA12k_Nov2014_V7 Probe Production
- Production of Padlock Probes targeting constitutive exon cDNA (V7 primers) of Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
Production PCR
- 10nM 1st round amplicons
- 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 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:011214 CA12kNov14 ProductionPCR V7.JPG
EtOH Precipitation
- 6 15-ml tubes (with 8 wells of PCR product each) 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 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 6 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
- Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~290ul of V7 probes: 93.2 ng/ul => ~27 ug
Lambda Exonuclease Digestion
- Divide each into 3 pcr tubes of ~100ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 100 |
10X Lambda Exo Buffer | 12.5 |
Lambda Exonuclease | 12.5 |
Total | 125 |
- Incubated at 37C for 1hr
- Purified with 3 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 62.9ng/ul x 120ul = 7.5ug (56% yield)
Remove Amplification Adapters
USER
- Save 2ul for gel check later
- Split into 2 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 59 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 8 |
Total | 80 |
- Incubate at 37C for 1.5 hours
DpnII
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 2 |
100uM RE-DpnII_V7 guide oligo | 5 |
H2O | 8 |
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight