Daniel:Notebook/ComboLock/2017-3-24: Difference between revisions
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<li>Mix two of the the following reaction(s)</li> | <li>Mix two of the the following reaction(s)</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> | <li>1 uL Padlock oligo (10 uM)</li> | ||
<li> | <li>4 uL CutSmart Buffer (10X)</li> | ||
<li> | <li>4 uL rSAP enzyme</li> | ||
<li> | <li>31 uL nfH2O</li></ol> | ||
<li>Incubate at 37C for | <li>Incubate at 37C for 1 hour</li> | ||
<li>Incubate at 65 for 5 minutes</li></ol> | <li>Incubate at 65 for 5 minutes</li></ol> | ||
<li>Ligation</li> | <li>Ligation</li> | ||
Line 28: | Line 28: | ||
| width="220" height="36" | Reagent | | width="220" height="36" | Reagent | ||
| width="100" | Stock Conc | | width="100" | Stock Conc | ||
| width=" | | width="200" | Final Conc./Amount | ||
| width="106" | uL added | | width="106" | uL added | ||
Line 39: | Line 39: | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Dephosphorylated Circ Latch (Step 1) | | height="15" valign="bottom" | Dephosphorylated Circ Latch (Step 1) | ||
| align="center" | | | align="center" | 250 nM | ||
| align="center" | | | align="center" | 1 pmol *This is less than the others | ||
| align="center" align="center" | | | align="center" align="center" | 4 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Padlock 0601 oligo | | height="15" valign="bottom" | Padlock 0601 oligo | ||
| align="center" | | | align="center" | 10 uM | ||
| align="center" | 10 pmol | | align="center" | 10 pmol | ||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
Line 51: | Line 51: | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | C4 Positive Control oligo | | height="15" valign="bottom" | C4 Positive Control oligo | ||
| align="center" | | | align="center" | 10 uM | ||
| align="center" | 10 pmol | | align="center" | 10 pmol | ||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
Line 57: | Line 57: | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | C2 positive Control oligo | | height="15" valign="bottom" | C2 positive Control oligo | ||
| align="center" | | | align="center" | 10 uM | ||
| align="center" | 10 pmol | | align="center" | 10 pmol | ||
| align="center" align="center" | 1 | | align="center" align="center" | 1 | ||
Line 71: | Line 71: | ||
| align="center" | NA | | align="center" | NA | ||
| align="center" | NA | | align="center" | NA | ||
| align="center" align="center" | | | align="center" align="center" | 8 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
Line 92: | Line 92: | ||
<li>Heat kill enzyme by incubating at 65C for 15 minutes</li> | <li>Heat kill enzyme by incubating at 65C for 15 minutes</li> | ||
<li>Add 20 uL nfH2O</li></ol> | <li>Add 20 uL nfH2O</li></ol> | ||
Continued [[Daniel:Notebook/ComboLock/2017-3-25|tomorrow]] | |||
[[Category:ComboLock]] [[Category: | [[Category:ComboLock]] [[Category:20170322]] |
Latest revision as of 18:01, 29 March 2017
Linear Product Production[edit]
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[edit]
- Dephosphorylation (Padlock Oligo)
- Mix two of the the following reaction(s)
- 1 uL Padlock oligo (10 uM)
- 4 uL CutSmart Buffer (10X)
- 4 uL rSAP enzyme
- 31 uL nfH2O
- Incubate at 37C for 1 hour
- 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
Reagent | Stock Conc | Final Conc./Amount | uL added |
T4 Ligase Buffer | 5X | 1X | 4 |
Dephosphorylated Circ Latch (Step 1) | 250 nM | 1 pmol *This is less than the others | 4 |
Padlock 0601 oligo | 10 uM | 10 pmol | 1 |
C4 Positive Control oligo | 10 uM | 10 pmol | 1 |
C2 positive Control oligo | 10 uM | 10 pmol | 1 |
T4 DNA Ligase | 5 U/uL | 5 U | 1 |
nfH2O | NA | NA | 8 |
Total | 20 |
Continued tomorrow