Matt:LabNotes/2014-8-15: Difference between revisions
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*Incubated at 37C for overnight (~15 hrs) | *Incubated at 37C for overnight (~15 hrs) | ||
===TBU Gel Check=== | |||
*Combine the 4 tubes | |||
*2ul Sample + 2ul 2X Urea Loading Dye | |||
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye | |||
*Digested sample | |||
*Undigested sample: 10nM 1st round amplicon | |||
[[]] | |||
*Brightest band below 200bp as expected | |||
===Column Purification=== | |||
*Used Zymo ssDNA/RNA columns | |||
*Total Eluted: 124 ul | |||
*Nanodrop quant: ng/ul | |||
*Yield: ng/ul x ul = ug | |||
<!-- | |||
===PAGE Size Selection=== | |||
*Run 6 gels of ~2.8ug ssDNA in each | |||
**Corresponds to 20.7ul in each gel | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | |||
| align="center" style="background:#f0f0f0;"|'''6X Volume''' | |||
|- | |||
| Enzyme digested probes||20.7||124 | |||
|- | |||
| TBE-Urea Buffer 2X||50||300 | |||
|- | |||
| Water||29.3||176 | |||
|- | |||
| Total||100||600 | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''6X Volume''' | |||
|- | |||
| Low Mass Ladder||6 | |||
|- | |||
| TBE-Urea Buffer 2X||30 | |||
|- | |||
| H2O||24 | |||
|- | |||
| Total||60 | |||
|} | |||
*Ran at 220V for 30min | |||
*Cut out brightest band below 200bp | |||
===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 12 Nanosep column 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 (~450ul per tube) | |||
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2 | |||
*Vortexed and placed the 12 tubes at -80C for 3 hrs | |||
*Spun 12 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 let dry in hood for 10min | |||
*Resuspend each tube with 10ul (120ul total) | |||
===TBU Gel Quantification=== | |||
*Diluted Agi26k0gap 10X | |||
**2ul Agi26k0gap + 8ul H2O + 10ul 2X TBE-Urea buffer | |||
*Made DNA Low Mass Ladder with 0.5, 1, and 2 ul | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Lane''' | |||
| align="center" style="background:#f0f0f0;"|'''1''' | |||
| align="center" style="background:#f0f0f0;"|'''2''' | |||
| align="center" style="background:#f0f0f0;"|'''3''' | |||
| align="center" style="background:#f0f0f0;"|'''4''' | |||
| align="center" style="background:#f0f0f0;"|'''5''' | |||
| align="center" style="background:#f0f0f0;"|'''6''' | |||
| align="center" style="background:#f0f0f0;"|'''7''' | |||
|- | |||
| Sample||0.5ul DNA Ladder||1ul DNA Ladder||2ul Sample||4ul Sample||6ul Sample||8ul Sample||2ul DNA Ladder | |||
|} | |||
[[File:2014-08-08_Agi26k0gapGelQuant.jpg]] | |||
*[[Media:2014-08-08_Agi26k0gapTBUGelQuantAnalysis.xlsx.xlsx | Quantification Analysis]] | |||
*Agi26k0gap Concentration: 38.32 ng/ul | |||
*Total Mass: 38.32 ng/ul * 120ul = 4.60 ug | |||
*Total Amount: 4.60ug /(193nt*325 Da/nt) = 73.3 pmole (611nM)--> |
Revision as of 00:17, 18 August 2014
Agi26k0gap Probe Production
Production PCR
- Make Master Mix for 51 well (7 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:2014-08-14 Agi26k0gapProductionPCR.JPG
EtOH Precipitation
- 6 15-ml tubes (with 8.5 wells of PCR product each) for 0gap
- 850ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 85ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3000rpm at 4C for 30min
- 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:
- ~225ul of Agi26k0gap probes: 317.5 ng/ul => 71.4 ug
Lambda Exonuclease Digestion
- Divide into seven pcr tubes of 32ul with total amplicon of ~10ug each
- Seventh tube had 33ul of amplicon
Components | Volume |
Amplicon | 32 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 48 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 89.0ng/ul x 255ul = 22.7ug (63.6% yield)
Removal of Amplification Adapters
Components | Volume |
ss-amplicon | 63.5 |
USER | 5 |
H2O | 11.5 |
total | 80 |
*~5ug ssDNA per tube
- Incubated at 37C for 2 hours
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 10 |
100uM RE-DpnII guide oligo | 5 |
RE-DpnII guide oligo: 0-gap: RE DpnII V6
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for 3 hr
- Added 100ul of the following
Components | Volume |
10X DpnII Buffer | 10 |
USER | 5 |
H2O | 85 |
Total | 100 |
- Incubated at 37C for overnight (~15 hrs)
TBU Gel Check
- Combine the 4 tubes
- 2ul Sample + 2ul 2X Urea Loading Dye
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
- Digested sample
- Undigested sample: 10nM 1st round amplicon
[[]]
- Brightest band below 200bp as expected
Column Purification
- Used Zymo ssDNA/RNA columns
- Total Eluted: 124 ul
- Nanodrop quant: ng/ul
- Yield: ng/ul x ul = ug