Matt:LabNotes/2016-7-26: Difference between revisions
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>Mzcai |
>Mzcai m (→Qubit ssDNA) |
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(3 intermediate revisions by the same user not shown) | |||
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===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
* | *Also include probes from [[Matt:LabNotes/2016-6-7]] | ||
* | [[File:2016-07-28_TB12kApr2016_V4_ProbeProduction_GelCheck.jpg|350px]] | ||
*You can see the 3 bands at the top | |||
*The full digested band is the brightest (third from top) and also kind of blurry due to amount | |||
*Run 6 gels for 6/7/2016 batch | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''6X Volume''' | |||
|- | |||
| V4 Probes||95 | |||
|- | |||
| TBE-Urea Buffer 2X||95 | |||
|} | |||
*Run 9 gels for this batch (wanted to do 10 but not enough gels) | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
Line 123: | Line 136: | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''15X Volume''' | ||
|- | |- | ||
| Low Mass Ladder|| | | Low Mass Ladder||15 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||75 | ||
|- | |- | ||
| H2O|| | | H2O||60 | ||
|- | |- | ||
| Total|| | | Total||150 | ||
|} | |} | ||
*200V for 40min | |||
*No gel images because too many gels | |||
**Bands looked good, could see '''very''' faint band of ~200bp and a less faint band below that and finally the completely digested probe band was brightest below that | |||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*Put cut out gel in | *Put cut out gel in 30 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT. | ||
*Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. | *Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. | ||
*Added 450 ul of 1X TE buffer to each | *Added 450 ul of 1X TE buffer to each | ||
*Vortexed for 60min at 37 C in incubator | *Vortexed for 60min 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 | ||
*Transferred the clear spnt. to | *Transferred the clear spnt. to Nanosep columns and centrifuged at 15,000 rpm for 3 min | ||
*Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | *Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | ||
*Transferred spnt to fresh 1.5 mL tube | *Transferred spnt to fresh 1.5 mL tube | ||
*Precipitated in | *Precipitated in 30 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2 | ||
*Vortexed and placed the | *Vortexed and placed the 30 tubes at -80C for over the weekend | ||
*Spun | *Spun 30 tubes at 10,000rpm at 4C for 30min | ||
*Discard supernatant and add 750ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min | *Discard supernatant and add 750ul cold 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 | ||
Line 151: | Line 167: | ||
===Qubit ssDNA=== | ===Qubit ssDNA=== | ||
22.7ng/ul x 300ul = 6,810ng<br> | |||
22.7ng/ul / (155nt x 303.7Da/nt + 79Da) = 478nM |
Latest revision as of 19:01, 2 August 2016
TB12k_Apr2016 V4 Probe Preparation[edit]
Production PCR[edit]
- Make 10ml V4 master mix for 96 wells
Components | Volume (1X) | Volume (100X) |
First round amplicon TB12k_Apr2016_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 | 10,000 |
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold
- Did in regular thermocyclers
EtOH Precipitation[edit]
- 8 15-ml tubes (with 12 wells (1.5 strips) of PCR product each) for
- 1,200ul PCR product
- 3000ul 100% EtOH
- 4ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 30 min
- 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 8 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[edit]
- 8 columns elute 50ul each
- Nanodrop: 374.4 ng/uL x 400uL = 149.76ug
Lambda Exo[edit]
- Divide into 8 pcr tubes of 100ul (ideally should be <10ug each)
Components | Volume |
Amplicon | 50 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 30 |
Total | 100 |
- Incubated at 37C for 3hr (normally 1hr)
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 167.0ng/ul x 320ul = 53.44ug
Remove Amplification Adapters[edit]
USER[edit]
- Split into 6 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 53.3 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 13.7 |
Total | 80 |
- Incubate at 37C for 2.5 hours
DpnII[edit]
- 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)
Zymo Column Purification[edit]
- Eluted 20ul each column (100ul total)
- Nanodrop
- 229.0 ng/ul x 140ul = 32.06 ug (59% yield)
PAGE Size Selection[edit]
- Also include probes from Matt:LabNotes/2016-6-7
File:2016-07-28 TB12kApr2016 V4 ProbeProduction GelCheck.jpg
- You can see the 3 bands at the top
- The full digested band is the brightest (third from top) and also kind of blurry due to amount
- Run 6 gels for 6/7/2016 batch
Components | 6X Volume |
V4 Probes | 95 |
TBE-Urea Buffer 2X | 95 |
- Run 9 gels for this batch (wanted to do 10 but not enough gels)
Components | 9X Volume |
V4 Probes | 135 |
TBE-Urea Buffer 2X | 135 |
Components | 15X Volume |
Low Mass Ladder | 15 |
TBE-Urea Buffer 2X | 75 |
H2O | 60 |
Total | 150 |
- 200V for 40min
- No gel images because too many gels
- Bands looked good, could see very faint band of ~200bp and a less faint band below that and finally the completely digested probe band was brightest below that
EtOH Precipitation[edit]
- Put cut out gel in 30 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
- Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
- Added 450 ul of 1X TE buffer to each
- Vortexed for 60min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to Nanosep columns and centrifuged at 15,000 rpm for 3 min
- Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transferred spnt to fresh 1.5 mL tube
- Precipitated in 30 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 30 tubes at -80C for over the weekend
- Spun 30 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 750ul cold 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 and combine
Qubit ssDNA[edit]
22.7ng/ul x 300ul = 6,810ng
22.7ng/ul / (155nt x 303.7Da/nt + 79Da) = 478nM