Daniel:Notebook/ComboLock/2017-3-24: Difference between revisions
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>Djacobse (Created page with "=RCA Testing (Circular Product)= Back to Calendar This experiment is to test the effectiveness of RCA so that I can use the most efficient meth...") |
>Djacobse |
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= | =Linear Product Production= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the [[Daniel:Notebook/ComboLock/2017-3-17|T4 experiments]] and the [[Daniel:Notebook/ComboLock/2017-3-22|circular product production]]. I will be using that circular product to analyze the effectiveness of RCA. | This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the [[Daniel:Notebook/ComboLock/2017-3-17|T4 experiments]] and the [[Daniel:Notebook/ComboLock/2017-3-22|circular product production]]. I will be using that circular product to analyze the effectiveness of RCA. | ||
However, as mentioned [[Daniel:Notebook/ComboLock/2017-3-23|yesterday]], first I want to make a linear product for an effective negative control. After reviewing the [https://www.neb.com/protocols/2013/06/10/protocol-for-dephosphorylation-of-5-ends-of-dna-m0371 recommended protocol] from NEB, it looks like I should use approximately 1 pmol of product per unit (uL) of enzyme I add. Yesterday, I added 4 uL of 100 uM circular product. For those of you who can do math: that's 400 pmol. Might have been a little too much. Modified protocol below will account for this. | |||
==Protocol== | ==Protocol== | ||
<ol> | |||
<li>Dephosphorylation (Padlock Oligo)</li> | |||
<ol type="A"> | |||
<li>Mix two of the the following reaction(s)</li> | |||
<ol type="a"> | |||
<li>2 uL Padlock oligo (10 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" | 2 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" | 10 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" | 10 pmol | |||
| align="center" align="center" | 1 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | C2 positive Control oligo | |||
| align="center" | 100 uM | |||
| align="center" | 10 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> | |||
[[Category:ComboLock]] [[Category:20170324]] | [[Category:ComboLock]] [[Category:20170324]] |
Revision as of 21:36, 24 March 2017
Linear Product Production
This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the T4 experiments and the circular product production. I will be using that circular product to analyze the effectiveness of RCA.
However, as mentioned yesterday, first I want to make a linear product for an effective negative control. After reviewing the recommended protocol from NEB, it looks like I should use approximately 1 pmol of product per unit (uL) of enzyme I add. Yesterday, I added 4 uL of 100 uM circular product. For those of you who can do math: that's 400 pmol. Might have been a little too much. Modified protocol below will account for this.
Protocol
- Dephosphorylation (Padlock Oligo)
- Mix two of the the following reaction(s)
- 2 uL Padlock oligo (10 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) | 2 uM | 100 pmol | 5 |
Padlock 0601 oligo | 100 uM | 10 pmol | 1 |
C4 Positive Control oligo | 100 uM | 10 pmol | 1 |
C2 positive Control oligo | 100 uM | 10 pmol | 1 |
T4 DNA Ligase | 5 U/uL | 5 U | 1 |
nfH2O | NA | NA | 6 |
Total | 20 |