Matt:LabNotes/2016-11-10: Difference between revisions
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>Mzcai m (→Lambda Exo) |
>Mzcai m (→DpnII) |
||
Line 101: | Line 101: | ||
*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 (~12hrs) | ||
===Zymo Column Purification=== | ===Zymo Column Purification=== | ||
*Eluted 20ul each column (180ul total) | *Eluted 20ul each column (180ul total) | ||
*Nanodrop | *Nanodrop | ||
** | **80.1 ng/ul x 180ul = 14.4ug (34% yield lower than normal) | ||
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===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*Run 2 gels | *Run 2 gels |
Revision as of 20:25, 14 November 2016
CA12k_Oct2016 V4 Probe Preparation
Production PCR
- 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
- 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
- 16 columns elute 50ul each
- Nanodrop: 170.3 ng/uL x 800uL = 136.24ug
Lambda Exo
- 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
USER
- 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
- 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
- Eluted 20ul each column (180ul total)
- Nanodrop
- 80.1 ng/ul x 180ul = 14.4ug (34% yield lower than normal)