Matt:LabNotes/2016-6-7: Difference between revisions
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>Mzcai m (→DpnII) |
>Mzcai |
||
Line 108: | Line 108: | ||
*Nanodrop | *Nanodrop | ||
**193.7 ng/ul x 100ul = 19.37 ug (% yield) | **193.7 ng/ul x 100ul = 19.37 ug (% yield) | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*Run | *Run 6 gels | ||
*200V for 40min | *200V for 40min | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''6X Volume''' | ||
|- | |- | ||
| V4 Probes||95 | | V4 Probes||95 | ||
Line 124: | Line 124: | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''6X Volume''' | ||
|- | |- | ||
| Low Mass Ladder|| | | Low Mass Ladder||6 | ||
|- | |- | ||
| TBE-Urea Buffer 2X|| | | TBE-Urea Buffer 2X||30 | ||
|- | |- | ||
| H2O|| | | H2O||24 | ||
|- | |- | ||
| Total|| | | Total||60 | ||
|} | |} | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*Put cut out gel in | *Put cut out gel in 12 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 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min | *Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min |
Revision as of 23:43, 28 July 2016
TB12k_Apr2016 V4 Probe Preparation
Production PCR
- Make 2X V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon TB12k_Apr2016_V4 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V4U (100uM) | 0.4 | 20 |
AP2V4 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold
- No curve because 100ul reaction volume only works in Biorad PCR machines
EtOH Precipitation
- 8 15-ml tubes (with 24 wells (3 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 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 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
- 8 columns elute 50ul each
- Nanodrop: 313.6 ng/uL x 400uL = 125.44ug
Lambda Exo
- 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 2hr (normally 1hr)
- Purified with 8 Zymo ssDNA/RNA columns
- Dropped one column so only 7
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 118.7ng/ul x 280ul = 33.24ug
Remove Amplification Adapters
USER
- Split into 5 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 56 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 11 |
Total | 80 |
- Incubate at 37C for 2.5 hours
DpnII
- 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
- Eluted 20ul each column (100ul total)
- Nanodrop
- 193.7 ng/ul x 100ul = 19.37 ug (% yield)
PAGE Size Selection
- Run 6 gels
- 200V for 40min
Components | 6X Volume |
V4 Probes | 95 |
TBE-Urea Buffer 2X | 95 |
Components | 6X Volume |
Low Mass Ladder | 6 |
TBE-Urea Buffer 2X | 30 |
H2O | 24 |
Total | 60 |
EtOH Precipitation
- Put cut out gel in 12 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