Matt:LabNotes/2014-8-15: Difference between revisions
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*Yield: 50.8 ng/ul x 260 ul = 13.2 ug | *Yield: 50.8 ng/ul x 260 ul = 13.2 ug | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*On 8/26/14 | |||
*Run 4 gels of ~3.3ug ssDNA in each | *Run 4 gels of ~3.3ug ssDNA in each | ||
**Corresponds to 65ul in each gel | **Corresponds to 65ul in each gel | ||
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*Cut out brightest band below 200bp | *Cut out brightest band below 200bp | ||
[[File:2014-08-26_Agi26k0gapSizeSelectionGel1.jpg]] | |||
[[File:2014-08-26_Agi26k0gapSizeSelectionGel2.jpg]] | |||
[[File:2014-08-26_Agi26k0gapSizeSelectionGel3.jpg]] | |||
[[File:2014-08-26_Agi26k0gapSizeSelectionGel4.jpg]] | |||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
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*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 8 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 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) | *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 | *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2 | ||
*Vortexed and placed the | *Vortexed and placed the 8 tubes at -80C for overnight | ||
*Spun | *Spun 8 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 | ||
*Discard supernatant and let dry in hood for 10min | *Discard supernatant and let dry in hood for 10min | ||
*Resuspend each tube with 10ul ( | *Resuspend each tube with 10ul (70ul total) | ||
===TBU Gel Quantification=== | ===TBU Gel Quantification=== | ||
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| Sample||0.5ul DNA Ladder||1ul DNA Ladder||2ul Sample||4ul Sample||6ul Sample||8ul Sample||2ul DNA Ladder | | Sample||0.5ul DNA Ladder||1ul DNA Ladder||2ul Sample||4ul Sample||6ul Sample||8ul Sample||2ul DNA Ladder | ||
|} | |} | ||
[[File:2014-08- | [[File:2014-08-27_Agi26k0gapGelQuant.jpg]] | ||
*[[Media:2014-08- | *[[Media:2014-08-27_Agi26k0gapGelQuant.xlsx | Quantification Analysis]] | ||
*Agi26k0gap Concentration: | *Agi26k0gap Concentration: 82.4 ng/ul | ||
*Total Mass: | *Total Mass: 82.4 ng/ul * 70ul = 5.77 ug | ||
*Total Amount: | *Total Amount: 5.77 ug /(193nt*325 Da/nt) = 92 pmole (1.314 uM) |
Latest revision as of 19:58, 2 September 2014
Agi26k0gap Probe Production[edit]
Production PCR[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
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[edit]
- 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
File:2014-08-17 Agi26k0gapDigestCheck.jpg
- Brightest band below 200bp as expected
Column Purification[edit]
- Used Zymo ssDNA/RNA columns
- Total Eluted: 260 ul
- Nanodrop quant: 50.8 ng/ul
- Yield: 50.8 ng/ul x 260 ul = 13.2 ug
PAGE Size Selection[edit]
- On 8/26/14
- Run 4 gels of ~3.3ug ssDNA in each
- Corresponds to 65ul in each gel
Components | 1X Volume | 4X Volume |
Enzyme digested probes | 65 | 260 |
TBE-Urea Buffer 2X | 65 | 260 |
Water | 0 | 0 |
Total | 130 | 520 |
Components | 4X Volume |
Low Mass Ladder | 4 |
TBE-Urea Buffer 2X | 20 |
H2O | 16 |
Total | 40 |
- Ran at 220V for 30min
- Cut out brightest band below 200bp
File:2014-08-26 Agi26k0gapSizeSelectionGel1.jpg File:2014-08-26 Agi26k0gapSizeSelectionGel2.jpg File:2014-08-26 Agi26k0gapSizeSelectionGel3.jpg File:2014-08-26 Agi26k0gapSizeSelectionGel4.jpg
EtOH Precipitation[edit]
- 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 8 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 8 tubes at -80C for overnight
- Spun 8 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 (70ul total)
TBU Gel Quantification[edit]
- Diluted Agi26k0gap 10X
- 2ul Agi26k0gap + 8ul H2O + 10ul 2X TBE-Urea buffer
- Made DNA Low Mass Ladder with 0.5, 1, and 2 ul
Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
Sample | 0.5ul DNA Ladder | 1ul DNA Ladder | 2ul Sample | 4ul Sample | 6ul Sample | 8ul Sample | 2ul DNA Ladder |
File:2014-08-27 Agi26k0gapGelQuant.jpg
- Agi26k0gap Concentration: 82.4 ng/ul
- Total Mass: 82.4 ng/ul * 70ul = 5.77 ug
- Total Amount: 5.77 ug /(193nt*325 Da/nt) = 92 pmole (1.314 uM)