Daniel:Notebook/ComboLock/2017-3-23: Difference between revisions
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|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Dephosphorylated Latch | | height="15" valign="bottom" | Dephosphorylated Circ Latch (Step 1) | ||
| align="center" | | | align="center" | 25 uM | ||
| align="center" | 100 pmol | | align="center" | 100 pmol | ||
| align="center" align="center" | | | align="center" align="center" | 4 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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| align="center" | NA | | align="center" | NA | ||
| align="center" | NA | | align="center" | NA | ||
| align="center" align="center" | | | align="center" align="center" | 7 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" |
Revision as of 22:55, 23 March 2017
Circular Product Production (Started Yesterday)
Protocol
- qPCR
- Make qPCR master mix according to following recipes
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170323-CPProduction.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
- Qubit Measurement
- Add 995 uL buffer and 5 uL ssdye to tube
- Add 190 uL buffer and 10 uL correct standard to standard tubes
- Add 199 uL buffer and 1 uL sample to sample tubes
- Let sit 5-10 minutes in the dark
- Measure in Qubit
- TBE Gel
- Mix 120 uL TBE and 40 uL 6X dye
- Aliquot 10 uL per sample on to parafilm
- Add 2 uL sample or 1.5 uL ladder to aliquots
- Mix and add 10 uL per lane to gel
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc
Reagent | 1X Rxn | MM-Amp (6.2X) | MM-Prod (7.2X) |
Primer Pair | NA | AmpF/AmpR | p24/p12RC |
Forward Primer (10 uM) | 1 | 6.2 | 7.2 |
Forward Primer (10 uM) | 1 | 6.2 | 7.2 |
Kapa SYBR Fast Master Mix (2X) | 25 | 155 | 180 |
nfH2O | 21 | 130.2 | 151.2 |
Total | 48 | 297.6 | 345.6 |
Results
- 20170323-qPCR-CPProd.png
qPCR curves
- 2017-03-23-CPProd-qPCR-CPTarget.png
Circular Product target (104bp)
- 2017-03-23-CPProd-qPCR-AmpF-AmpR.png
AmpF/AmpR (221 bp)
Use the AmpF/AmpR gel as a reference point in the future. Also, positive controls for qPCR and the like should be done at a 1:10 dilution of the original. The original concentration, via Qubit is:
Qubit | Dil | Meas | Amt | Average (ng/uL) | uM |
x1 | 1 | 118 | 118 | ||
x2 | 5 | 18.3 | 91.5 | ||
x3 | 10 | 8.94 | 89.4 | 99.6 | 1.61 |
Linear Product Production
This is the next step in planning my RCA experiment. This will serve as a negative control for the experiment, since no circular product should be formed.
Protocol
- Dephosphorylation (Bolt Oligo)
- Mix the following reaction
- 5 uL Circ Latch oligo (100 uM)
- 2 uL CutSmart Buffer (10X)
- 2 uL rSAP enzyme
- 16 uL nfH2O
- Incubate at 37C for 30 minutes
- Incubate at 65 for 5 minutes
- Ligation
- Make reaction mixture according to table; Do not add ligase yet
- Use following thermocycler program
- Heat mixture to 95C for 5 min
- Lower temp by 0.2C per second to 55C
- Hold mixture at 55C for 15 minutes
- Lower temp to 20C by reducing 0.2C per second
- Hold at 20C
- Add 1 uL T4 DNA ligase
- Incubate at 20C for 30 minutes
- Heat kill enzyme by incubating at 65C for 15 minutes
- Add 20 uL nfH2O
- TBE gel
- Mix 64 uL TBE and 16 uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot Gel lanes
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 Ligase Buffer | 5X | 1X | 4 |
Dephosphorylated Circ Latch (Step 1) | 25 uM | 100 pmol | 4 |
Padlock 0601 oligo | 100 uM | 100 pmol | 1 |
C4 Positive Control oligo | 100 uM | 100 pmol | 1 |
C2 positive Control oligo | 100 uM | 100 pmol | 1 |
T4 DNA Ligase | 5 U/uL | 5 U | 1 |
nfH2O | NA | NA | 7 |
Total | 20 |