Matt:LabNotes/2016-5-25: Difference between revisions
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>Mzcai m (→Lambda Exo) |
>Mzcai m (→Lambda Exo) |
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Line 177: | Line 177: | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
** | **91.1ng/ul x 320ul = 29.15ug (% yield) |
Revision as of 20:47, 1 June 2016
TB12k_Apr2016 Probe Preparation
- Probe Design
- Received 195-mer oligos 5/25/26
- Name: TB12k_Apr2016_DARTFISH
- Amount: 341ng
- Physical State: Solid
- Calculate MW of 195 nt probe = 195nt * 303.7Da/nt + 79Da = 59,300.5 g/mol
- 341ng -> 5.75pmol
Resuspend and Aliquot
- Resuspend in 100ul (57.5nM)
- Pipetted 20X to mix
- Made aliquots of 40, 40, 10, and 10 into 0.5ml Lo-Bind tubes
Serial Dilution 1000X
- Take 1ul from a 10ul aliquot and add 9ul H2O (10X Dilution)
- Take 5ul and add 45ul H2O (100X Dilution)
- Take 20ul and add 180ul H2O (1000X Dilution)
Expansion PCR Test
- NTC
Component | Volume |
Seed oligo | 0 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 10.5 |
Total | 25 |
- Positive Control
Component | Volume |
CA12kNov2014_V4 1st rnd Oligos (10nM) | 2.5 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 8 |
Total | 25 |
- 1,000X Dilution
Component | Volume |
Seed oligo (100X Dilution) | 2.5 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 8 |
Total | 25 |
- 10,000X Dilution
Component | Volume |
Seed oligo (1000X Dilution) | 2.5 |
F/R V4 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 8 |
Total | 25 |
Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold
File:20160526 TB12k ExpansionPCRtest.JPG
- 1,000X final dilution was in the sweet spot for number of cycles (15-20)
- Use that concentration and do 19 cycles
Expansion PCR
Component | Volume |
Seed oligo (100X Dilution) | 15 |
F/R V4 Primer Mix (10uM) | 12 |
2X Kapa MM | 75 |
H2O | 48 |
Total | 150 |
- 50ul per PCR tube
Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold
- Purified with 2 Qiagen PCR columns (75ul PCR product each)
- Elute with 50ul H2O
- Quantify with Qubit dsDNA:
- 10.1ng/ul / (195bp*607.4Da/bp+157.9Da) = 85.16 nmol/L
- Dilute to 10nM
- 85.16nM x 99ul = 10nM x 843ul
- Add 744ul H2O
Production PCR
- 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 22 -> 72C 2min -> 15C hold
- Stopped after cycles
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 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
- 8 columns elute 50ul each
- Nanodrop: 287.3 ng/uL x 400uL = 114.9ug
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
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 91.1ng/ul x 320ul = 29.15ug (% yield)