Matt:LabNotes/2014-9-18: Difference between revisions
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===Qia Column Purification=== | ===Qia Column Purification=== | ||
*Purified 6 tubes of 100ul in 6 columns following Qiagen protocol | *Purified 6 tubes of 100ul in 6 columns following Qiagen protocol | ||
*Eluted each column with 40ul and combined all probes into one 1.5ml | *Eluted each column with 40ul and combined all probes into one 1.5ml | ||
*Measured concentration with Nanodrop: | *Measured concentration with Nanodrop: | ||
*~225ul of Agi26k0gap probes: | *~225ul of Agi26k0gap probes: 335.3 ng/ul => 75.4 ug | ||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide into | *Divide into eight pcr tubes of 29ul 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||29 | ||
|- | |- | ||
| 10X Lambda Exo Buffer||10 | | 10X Lambda Exo Buffer||10 | ||
Line 63: | Line 62: | ||
| Lambda Exonuclease||10 | | Lambda Exonuclease||10 | ||
|- | |- | ||
| H2O|| | | H2O||51 | ||
|- | |- | ||
| Total||100 | | Total||100 | ||
Line 72: | Line 71: | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | ** 67.8ng/ul x 310ul = 21.0ug | ||
===Removal of Amplification Adapters=== | ===Removal of Amplification Adapters=== | ||
*5 tubes | |||
{| {{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||62 | ||
|- | |- | ||
| USER||5 | | USER||5 | ||
|- | |- | ||
| H2O|| | | H2O||13 | ||
|- | |- | ||
| total||80 | | total||80 | ||
Line 103: | Line 102: | ||
*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 | |||
===TBU Gel Check=== | ===TBU Gel Check=== | ||
*Combine the | *Combine the 5 tubes | ||
*2ul Sample + 2ul 2X Urea Loading Dye | *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 | ||
*Digested sample | *Digested sample | ||
*Undigested sample: | *Undigested sample: 2ul prior to Lambda digest | ||
[[File:2014- | [[File:2014-09-24_Agi26k0gapDigestCheck.jpg|250px]] | ||
*Brightest band below 200bp as expected | *Brightest band below 200bp as expected | ||
===Column Purification=== | ===Column Purification=== | ||
*Used Zymo ssDNA/RNA columns | *Used Zymo ssDNA/RNA columns | ||
*Total Eluted: | *Total Eluted: 200 ul | ||
*Nanodrop quant: | *Nanodrop quant: 79.1 ng/ul | ||
*Yield: | *Yield: 79.1 ng/ul x 200 ul = 15.8 ug | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*Run 5 gels of ~3ug ssDNA in each | |||
*Run | **Corresponds to 40ul in each gel | ||
**Corresponds to | |||
{| {{table}} | {| {{table}} | ||
Line 146: | Line 130: | ||
| align="center" style="background:#f0f0f0;"|'''4X Volume''' | | align="center" style="background:#f0f0f0;"|'''4X Volume''' | ||
|- | |- | ||
| Enzyme digested probes|| | | Enzyme digested probes||40||200 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||40||200 | ||
|- | |- | ||
| Water||0||0 | | Water||0||0 | ||
|- | |- | ||
| Total|| | | Total||80||400 | ||
|} | |} | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''5X Volume''' | ||
|- | |- | ||
| Low Mass Ladder|| | | Low Mass Ladder||5 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||25 | ||
|- | |- | ||
| H2O|| | | H2O||20 | ||
|- | |- | ||
| Total|| | | Total||50 | ||
|} | |} | ||
*Ran at 220V for 30min | *Ran at 220V for 30min | ||
*Cut out brightest band below 200bp | *Cut out brightest band below 200bp | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
Line 182: | Line 161: | ||
*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 10 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 10 tubes at -80C for overnight | ||
*Spun | *Spun 10 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 (100ul total) | ||
===TBU Gel Quantification=== | ===TBU Gel Quantification=== | ||
Line 209: | Line 188: | ||
| 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: | [[File:20141001_Agi26k0gapGelQuant.jpg]] | ||
*[[Media: | *[[Media:20141001_Agi26k0gapGelQuant.xlsx | Quantification Analysis]] | ||
*Agi26k0gap Concentration: | *Agi26k0gap Concentration: 93.15 ng/ul | ||
*Total Mass: | *Total Mass: 93.15 ng/ul * 100ul = 9.3 ug | ||
*Total Amount: | *Total Amount: 9.3 ug /(193nt*325 Da/nt) = 148 pmole (1.483 uM) | ||
Latest revision as of 02:11, 2 October 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:20140919 Agi26k0gapProductionPCR.PNG
EtOH Precipitation[edit]
- 6 5-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: 335.3 ng/ul => 75.4 ug
Lambda Exonuclease Digestion[edit]
- Divide into eight pcr tubes of 29ul with total amplicon of ~10ug each
Components | Volume |
Amplicon | 29 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 51 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 67.8ng/ul x 310ul = 21.0ug
Removal of Amplification Adapters[edit]
- 5 tubes
Components | Volume |
ss-amplicon | 62 |
USER | 5 |
H2O | 13 |
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 overnight
TBU Gel Check[edit]
- Combine the 5 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: 2ul prior to Lambda digest
File:2014-09-24 Agi26k0gapDigestCheck.jpg
- Brightest band below 200bp as expected
Column Purification[edit]
- Used Zymo ssDNA/RNA columns
- Total Eluted: 200 ul
- Nanodrop quant: 79.1 ng/ul
- Yield: 79.1 ng/ul x 200 ul = 15.8 ug
PAGE Size Selection[edit]
- Run 5 gels of ~3ug ssDNA in each
- Corresponds to 40ul in each gel
Components | 1X Volume | 4X Volume |
Enzyme digested probes | 40 | 200 |
TBE-Urea Buffer 2X | 40 | 200 |
Water | 0 | 0 |
Total | 80 | 400 |
Components | 5X Volume |
Low Mass Ladder | 5 |
TBE-Urea Buffer 2X | 25 |
H2O | 20 |
Total | 50 |
- Ran at 220V for 30min
- Cut out brightest band below 200bp
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 10 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 10 tubes at -80C for overnight
- Spun 10 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 (100ul 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:20141001 Agi26k0gapGelQuant.jpg
- Agi26k0gap Concentration: 93.15 ng/ul
- Total Mass: 93.15 ng/ul * 100ul = 9.3 ug
- Total Amount: 9.3 ug /(193nt*325 Da/nt) = 148 pmole (1.483 uM)