Matt:LabNotes/2016-6-7
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TB12k_Apr2016 V4 Probe Preparation[edit]
Production PCR[edit]
- 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[edit]
- 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[edit]
- 8 columns elute 50ul each
- Nanodrop: 313.6 ng/uL x 400uL = 125.44ug
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 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[edit]
USER[edit]
- 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[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
- 193.7 ng/ul x 100ul = 19.37 ug (% yield)