Matt:LabNotes/2015-1-13: Difference between revisions
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*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 overnight | *Incubated at 37C for overnight (~15hrs) | ||
===TBU Gel: Digestion Check=== | ===TBU Gel: Digestion Check=== | ||
*Combine into 1 tube | *Combine into 1 tube | ||
*2ul | *2ul Sample + 2ul 2X Urea Loading Dye | ||
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye | *0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye | ||
[[File: | [[File:]] | ||
* | *Gel ran poorly for some reason but I don't have anymore undigested probe to compare to (unless I want to waste 5ul of 10nM 1st rnd amplicon) so I will just check the gel when I do size selection | ||
* | **The band to cut out should be 150bp so almost halfway between 200bp and 100bp ladder bands | ||
* | |||
===Zymo Column Purification=== | ===Zymo Column Purification=== | ||
*Eluted 30ul each column (60ul total) | |||
*Eluted 30ul each column ( | |||
*Nanodrop | *Nanodrop | ||
** | **41.7ng/ul x 60ul = 2.5ug (oddly low again like [[Matt:LabNotes/2015-1-5#Zymo_Column_Purification | last time]]) | ||
***This time 33% yield which is better than 25% yield last time | |||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*Run | *Run 1 gel for V7 | ||
**220V for 30min | **220V for 30min | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | | align="center" style="background:#f0f0f0;"|'''1X Volume''' | ||
|- | |- | ||
| V7 Probes|| | | V7 Probes||60 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||60 | ||
|} | |} | ||
Line 163: | Line 135: | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| Low Mass Ladder|| | | Low Mass Ladder||1 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||5 | ||
|- | |- | ||
| H2O|| | | H2O||4 | ||
|- | |- | ||
| Total|| | | Total||10 | ||
|} | |} | ||
[[File:]] | |||
[[File: | |||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
Line 186: | Line 152: | ||
*Vortexed for 60 min at 37 C in incubator | *Vortexed for 60 min at 37 C in incubator | ||
*Centrifuged at 15,000 rpm for 3 min at RT | *Centrifuged at 15,000 rpm for 3 min at RT | ||
*Transfered the clear spnt. to | *Transfered the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min | ||
*Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | *Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | ||
*Transfered spnt to fresh 1.5 mL tube (~400ul per tube) | *Transfered spnt to 2 fresh 1.5 mL tube (~400ul per tube) | ||
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 | *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 | ||
*Vortexed and placed the | *Vortexed and placed the 2 tubes at -80C for overnight<!-- | ||
*Spun 6 tubes at 10,000rpm at 4C for 30min | *Spun 6 tubes at 10,000rpm at 4C for 30min | ||
*Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min | *Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min |
Revision as of 02:49, 15 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 2.5 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 (~15hrs)
TBU Gel: Digestion Check
- Combine into 1 tube
- 2ul Sample + 2ul 2X Urea Loading Dye
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
[[File:]]
- Gel ran poorly for some reason but I don't have anymore undigested probe to compare to (unless I want to waste 5ul of 10nM 1st rnd amplicon) so I will just check the gel when I do size selection
- The band to cut out should be 150bp so almost halfway between 200bp and 100bp ladder bands
Zymo Column Purification
- Eluted 30ul each column (60ul total)
- Nanodrop
- 41.7ng/ul x 60ul = 2.5ug (oddly low again like last time)
- This time 33% yield which is better than 25% yield last time
- 41.7ng/ul x 60ul = 2.5ug (oddly low again like last time)
PAGE Size Selection
- Run 1 gel for V7
- 220V for 30min
Components | 1X Volume |
V7 Probes | 60 |
TBE-Urea Buffer 2X | 60 |
Components | Volume |
Low Mass Ladder | 1 |
TBE-Urea Buffer 2X | 5 |
H2O | 4 |
Total | 10 |
[[File:]]
EtOH Precipitation
- Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
- Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
- Added 900 ul of 1X TE buffer
- Vortexed for 60 min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transfered the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
- Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transfered spnt to 2 fresh 1.5 mL tube (~400ul per tube)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
- Vortexed and placed the 2 tubes at -80C for overnight