Daniel:Notebook/PosSequencing/2017-5-19
Jump to navigation
Jump to search
Double Stranded Probe Production[edit]
I need to make more of these.
Protocol[edit]
- qPCR
- Make three 8.2X (1X) master mix with the following reagents
- 8.2 (1) uL 10 uM AP1v4U
- 8.2 (1) uL 10 uM BiotinU
- 172.2 (21) uL nfH2O
- 205 (25) uL 2X SYBR Fast
- 16.4 (2) appropriate barcode oligo (100 pM)
- Add 50 uL master mix to 8 lanes per sample File:PlateLayout-20170519-BarcodeOligoProduction.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- Qiaquick Column Note: I will be pooling samples and double loading columns, yielding 4 samples per column (2 columns per barcode oligo, 6 columns total)
- Pool 2 samples and 500 uL PB (5X binding buffer)
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add another 600 uL pooled sample to columnand spin for 1 minute at 14000 rpm; discard flow through
- Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Add 35 uL nfH2O to column
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- Pool samples when finished; concentrate if desired
- TBE Gel
- Mix 48 uL TBE and 12 uL 6X dye
- Aliquot 10 uL mix to parafilm
- Add 2 uL sample or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to appropriate lane
- Run gel at 240V for 24 minutes
- Open gel and stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
- Qubit
- Mix 1194 uL dsDNA buffer and 6 uL dye
- Aliquot 190 uL to standard tubes and 199 uL to dye tubes
- Add 10 uL standard or 1 uL dye
- Vortex and spin down
- Let sit 5 minutes in the dark
- Measure in the Qubit
Results[edit]
Sample | Qubit Conc (ug/mL) | Amount (nM) | Total Amount (fmol) | Amplification (Over 40 amol) |
Barcode Oligo v1 | 20.1 | 324 | 2.6E+04 | 6.5E+05 |
Barcode Oligo v2 | 16.3 | 263 | 2.1E+04 | 5.3E+05 |
Barcode Oligo v3 | 18 | 290 | 2.3E+04 | 5.8E+05 |
- 20170519-qPCR-BarcodeOligoProduction.png
qPCR curves
Exonuclease Digestion Test[edit]
I'm going to use lambda exonuclease to digest the second strand (it can't digest the biotin strand since it has a 5' biotin)
- Phosphorylation
- Set up 2 reactions using the following table
- Incubate at 37C for 30 minutes
- Incubate at 65C for 20 minutes
- Lambda Exonuclease Digestion
- Prepare 2 reactions according to following table
- Incubate at 37C for 30 minutes
- Incubate at 80C for 10 minutes
- Hold at 12C
Reagent | Final Conc. | uL Added |
dsDNA (0.3 pmol/uL) | (< 300 pmol) | 10 |
Reaction Buffer 10X | 1X | 5 |
ATP (10 mM) | 1 mM | 5 |
T4 PNK (10 U/uL) | 10 Units | 1 |
nfH2O | NA | 29 |
Total | 50 |
Reagent | Final Conc. | uL Added |
Phosphorylated Mix | (<2 ug) | 20 |
Reaction Buffer 10X | 1X | 5 |
Lamda Exonuclease (10 U/uL) | 2 U/uL | 2 |
nfH2O | NA | 23 |
Total | 50 |
Continued tomorrow