Matt:LabNotes/2015-6-3: Difference between revisions
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*Total volume: 300ul | *Total volume: 300ul | ||
*Nanodrop quantification: 155.7 ng/ul -> 46.7 ug | *Nanodrop quantification: 155.7 ng/ul -> 46.7 ug | ||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide into 6 pcr tubes of | *Divide into 6 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|| | | 10X Lambda Exo Buffer||10 | ||
|- | |- | ||
| Lambda Exonuclease|| | | Lambda Exonuclease||10 | ||
|- | |- | ||
| Total|| | | H2O||30 | ||
|- | |||
| Total||100 | |||
|} | |} | ||
Line 89: | Line 69: | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **ng/ul x 240ul = ug (76% yield) | ||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
Line 127: | Line 107: | ||
*Added 5ul DpnII | *Added 5ul DpnII | ||
*Incubated at 37C for overnight (~15hrs) | *Incubated at 37C for overnight (~15hrs) | ||
<!-- | |||
===Zymo Column Purification=== | ===Zymo Column Purification=== | ||
*Eluted 30ul each column (115ul total) | *Eluted 30ul each column (115ul total) |
Revision as of 19:47, 4 June 2015
CA12k_Nov2014_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
Production PCR
- 10nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon CA12k_Nov14_V4 (10nM) | 0.2 | 20 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (100uM) | 0.4 | 40 |
H2O | 49 | 4900 |
Total | 100 | 10000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
QIAEX-II Purification
- Divide into 8 5mL tubes of 1200ul each
- Add 3600ul (3X volume) of Buffer QX1
- Vortex the QIAEXII for 30sec
- Add 30ul QIAEXII (30ul for every 10ug)
- Incubate at 50C while mixing for 10min
- Color should be yellow
- Centrifuge sample for 30sec
- Remove supernatant with a pipette
- Wash the pellet with 500ul Buffer QX1
- Resuspend the pellet by vortexing
- Transfer to 1.5ml tubes
- Centrifuge sample for 30sec and remove supernatant again with pipette
- Wash the pellet twice with 500ul Buffer PE
- Air-dry the pellet for 30min until the pellet turns white
- To elute, resuspend with 20ul H2O and vortexing
- Incubate at RT for 5min
- Centrifuge for 30sec and pipette out supernatant with sample
- Repeat 20ul H2O resuspension and incubation to improve yield 10-15%
- Total volume: 300ul
- Nanodrop quantification: 155.7 ng/ul -> 46.7 ug
Lambda Exonuclease Digestion
- Divide into 6 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 6 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- ng/ul x 240ul = ug (76% yield)
Remove Amplification Adapters
USER
- Split into 4 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 2.5 hours
DpnII
- 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)