Daniel:Notebook/ComboLock/2017-3-23: Difference between revisions
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===Results=== | ===Results=== | ||
<gallery perrow= | <gallery perrow=3 heights=300px widths=300px> | ||
File: | File:20170323-qPCR-CPProd.png|qPCR curves | ||
File:2017-03- | File:2017-03-23-CPProd-qPCR-CPTarget.png|Circular Product target (104bp) | ||
File:2017-03- | File:2017-03-23-CPProd-qPCR-AmpF-AmpR.png|AmpF/AmpR (221 bp) | ||
</gallery> | </gallery> | ||
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: | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#80FF00;font-size:12pt;font-weight:bold" align="center" | |||
| width="85" height="30" | Qubit | |||
| width="85" | Dil | |||
| width="105" | Meas | |||
| width="105" | Amt | |||
| width="105" | Average (ng/uL) | |||
| width="105" | uM | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | x1 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 118 | |||
| align="center" align="center" valign="bottom" | 118 | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | x2 | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 18.3 | |||
| align="center" align="center" valign="bottom" | 91.5 | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" align="center" valign="bottom" | | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | x3 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 8.94 | |||
| align="center" align="center" valign="bottom" | 89.4 | |||
| align="center" align="center" valign="bottom" | 99.6 | |||
| align="center" align="center" valign="bottom" | 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== | |||
<ol> | |||
<li>Dephosphorylation (Bolt Oligo)</li> | |||
<ol type="A"> | |||
<li>Mix the following reaction</li> | |||
<ol type="a"> | |||
<li>4 uL Circ Latch oligo (100 uM)</li> | |||
<li>2 uL CutSmart Buffer (10X)</li> | |||
<li>2 uL rSAP enzyme</li> | |||
<li>12 uL nfH2O</li></ol> | |||
<li>Incubate at 37C for 30 minutes</li> | |||
<li>Incubate at 65 for 5 minutes</li></ol> | |||
<li>Ligation</li> | |||
<ol type="A"> | |||
<li>Make reaction mixture according to table; '''Do not add ligase yet'''</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#66FFFF;font-size:12pt;font-weight:bold" align="center" | |||
| width="220" height="36" | Reagent | |||
| width="100" | Stock Conc | |||
| width="100" | Final Conc./Amount | |||
| width="106" | uL added | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | T4 Ligase Buffer | |||
| align="center" | 5X | |||
| align="center" | 1X | |||
| align="center" align="center" | 4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Dephosphorylated Circ Latch (Step 1) | |||
| align="center" | 20 uM | |||
| align="center" | 100 pmol | |||
| align="center" align="center" | 5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Padlock 0601 oligo | |||
| align="center" | 100 uM | |||
| align="center" | 100 pmol | |||
| align="center" align="center" | 1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | C4 Positive Control oligo | |||
| align="center" | 100 uM | |||
| align="center" | 100 pmol | |||
| align="center" align="center" | 1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | C2 positive Control oligo | |||
| align="center" | 100 uM | |||
| align="center" | 100 pmol | |||
| align="center" align="center" | 1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | T4 DNA Ligase | |||
| align="center" | 5 U/uL | |||
| align="center" | 5 U | |||
| align="center" align="center" | 1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" | NA | |||
| align="center" | NA | |||
| align="center" align="center" | 6 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Total | |||
| align="center" align="center" | | |||
| align="center" align="center" | | |||
|style="font-weight:bold" align="center" align="center" | 20 | |||
|} | |||
<li>Use following thermocycler program</li> | |||
<ol type="a"> | |||
<li>Heat mixture to 95C for 5 min</li> | |||
<li>Lower temp by 0.2C per second to 55C</li> | |||
<li>Hold mixture at 55C for 15 minutes</li> | |||
<li>Lower temp to 20C by reducing 0.2C per second</li> | |||
<li>Hold at 20C</li></ol> | |||
<li>Add 1 uL T4 DNA ligase</li> | |||
<li>Incubate at 20C for 30 minutes</li> | |||
<li>Heat kill enzyme by incubating at 65C for 15 minutes</li> | |||
<li>Add 20 uL nfH2O</li></ol> | |||
<li>TBE gel</li> | |||
<li>Mix 64 uL TBE and 16 uL 6X dye</li> | |||
<li>Aliquot 10 uL on to parafilm per sample/ladder</li> | |||
<li>Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot</li> | |||
[[Image:G|400px]] | |||
<li>Add 10 uL mix to gel lanes</li> | |||
<li>Run gel for 24 minutes at 230V</li> | |||
<li>Stain with 2 uL SYBR gold for 3 minutes</li> | |||
<li>Rinse and image in gel doc</li> | |||
</ol> | |||
[[Image:2017-03-24-LinearProductProduction.png|500px]] | |||
[[Category:ComboLock]] [[Category:20170322]] |
Latest revision as of 16:28, 24 March 2017
Circular Product Production (Started Yesterday)[edit]
Protocol[edit]
- 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[edit]
- 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[edit]
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[edit]
- Dephosphorylation (Bolt Oligo)
- Mix the following reaction
- 4 uL Circ Latch oligo (100 uM)
- 2 uL CutSmart Buffer (10X)
- 2 uL rSAP enzyme
- 12 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 File:G
- 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) | 20 uM | 100 pmol | 5 |
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 | 6 |
Total | 20 |