Daniel:Notebook/ComboLock/2017-3-24
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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 |