Matt:LabNotes/2016-11-10: Difference between revisions
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>Mzcai |
>Mzcai |
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*Discard supernatant and let dry in hood for 10min | *Discard supernatant and let dry in hood for 10min | ||
*Resuspend each tube with 10ul and combine | *Resuspend each tube with 10ul and combine | ||
===Qubit ssDNA=== | ===Qubit ssDNA=== | ||
7.79ng/ul x 60ul = 467.4ng | |||
7.79ng/ul / (157 x 303.7Da/nt + 79Da) = 160nM | |||
Latest revision as of 23:49, 15 November 2016
CA12k_Oct2016 V4 Probe Preparation[edit]
Production PCR[edit]
- V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Oct2016_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 |
- Did one plate - 96 wells 100ul each
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold 450px
EtOH Precipitation[edit]
- 16 5-ml tubes each with
- 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 16 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]
- 16 columns elute 50ul each
- Nanodrop: 170.3 ng/uL x 800uL = 136.24ug
Lambda Exo[edit]
- Divide into 14 pcr tubes of 100ul (ideally should be <10ug each)
Components | Volume |
Amplicon | 57 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 23 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 14 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 72.4ng/ul x 560ul = 40.54ug (60% yield)
Remove Amplification Adapters[edit]
USER[edit]
- Split into 9 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 62.2 |
USER | 5 |
10X DpnII Buffer | 8 |
H2O | 4.8 |
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 (~12hrs)
Zymo Column Purification[edit]
- Eluted 20ul each column (180ul total)
- Nanodrop
- 80.1 ng/ul x 180ul = 14.4ug (36% yield lower than normal)
PAGE Size Selection[edit]
- Run 3 gels (normally should be 5 gels to keep < 3ug per gel)
- 200V for 40min
Components | 2X Volume |
V4 Probes | 180 |
TBE-Urea Buffer 2X | 180 |
Components | 2X Volume |
Low Mass Ladder | 3 |
TBE-Urea Buffer 2X | 15 |
H2O | 12 |
Total | 30 |
EtOH Precipitation[edit]
- Put cut out gel in 6 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 2 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 6 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 6 tubes at -80C for 30min
- Spun 6 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]
7.79ng/ul x 60ul = 467.4ng 7.79ng/ul / (157 x 303.7Da/nt + 79Da) = 160nM