Daniel:Notebook/ComboLock/2016-12-9: Difference between revisions
>Djacobse |
>Djacobse |
||
Line 86: | Line 86: | ||
<li>Add 10 uL mix to each sample</li> | <li>Add 10 uL mix to each sample</li> | ||
<li>Incubate at 37C for 30 min with agitation</li> | <li>Incubate at 37C for 30 min with agitation</li> | ||
<li>Wash twice with 100 uL wash buffer</li> | |||
<li>Remove supernatant and resuspend in 20 uL 1X Amp ligase buffer</li> | |||
</ol> | |||
<li>Circularization</li> | <li>Circularization</li> | ||
<ol type="A"> | <ol type="A"> |
Revision as of 23:52, 9 December 2016
Mirror Probe Protocol
New Directions
This protocol covers a new method using ideas taken from Weibrecht et al New Biotechnology 2012 (Landegren lab). The main idea I see is that they almost never use polymerase, but prefer to instead use ligation events as the predominant form. Based on the results from the extended latch experiment, the RCA-based cell test, and the sequencing from the first circularization test, it seems the probes do not circularize correctly. Note the original orientation of the C probes with the 5' end being the genome matching region and the 3' end being the latch/padlock adapter. When subjected to polymerization, if the latch is not present this yields a product that follows the backbone of the second C probe. See image below for clarification.
- Original C probes, orientation and binding. C probes in red, padlock probe in green, polymerase-added bases in magenta, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.
- C Probes-Original.png
Original C probe orientation
- C Probes-BadCircularization.png
Polymerization without latch traces along the C2 probe
This actually makes quite a bit of sense since the probability of only the padlock binding is much higher than the probability of both the latch and padlock binding.
Mirror Probes
To address this problem, we now introduce the mirror C probes. These C probes have their 3' and 5' ends reversed such that the 3' end is the genome matching region and the 5' end is the adapter region. Notice that because of the new orientation a polymerase that has no latch to work off will fall off the second C probe, rendering it impossible to circularize (hopefully).
- Mirror C probes have the advantage of polymerase failing to trace them by mistake. Original C probes, orientation and binding. C probe in red, mirror probe in blue, padlock probe in green, polymerase-added bases in magenta, and template in black. Vertical line indicates 5' end, arrow indicates 3' end.
- C Probes-MirrorHybrid.png
Orientation of the mirror C probe (blue)
- C Probes-Mirror-Polymerization.png
Polymerase product "falls off" the 3' end of the mirror C probe and does not create a lengthy product
To this end I have ordered two control mirror C probes to test (for the positive control oligo).
Lock Oligos (Ligation-Based Templates)
I have also had a second idea based off the Landegren (Weibrecht) paper. The idea is to add a ligation event that will connect together the two C probes (via the latch). This will provide a stable template for the padlock probe that becomes latch independent. First, we look at just the latch binding.
Now I introduce the "Lock oligo", which is reverse complement to the latch oligo. Rather than incubating the latch and padlock at the same time, we incubate the lock and latch at the same time which ligate the C probes together. It then no longer matters if the latch remains bound to the adapter regions, the padlock has a contiguous template to work off.
- Modified latch-padlock system integrating the lock oligo. Lock/Latch binding is ligase based-only creating a solid block to read for polymerization. C probe in red, mirror C probe in blue, latch in dashed green, polymerization-added bases in magenta, lock oligo in brown, and template in black.
- Latch Binding.png
Latch binding event
- LockBinding.png
Lock binding event; the lock is RC to the region between adapters (in v1 latches, the barcode+UMI)
- LockBridge.png
Lock-C probe ligation produces a "bridge" for padlock circularization
- LockPadlockCircularization.png
Padlock now binds with a complete template and can circularize/ligate for further amplification
Experiment
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probe |
Protocol
- Template-Bead Binding
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- C Probe Hybridization
- Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation; start part 3 after incubation begins
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
- Latch and Padlock Hybridization
- Add 1 uL Latch0001 and 1 uL Padlock0201 to 8 uL wash buffer (per sample)
- Add 10 uL mix to each sample
- Incubate at 37C for 30 min with agitation
- Wash twice with 100 uL wash buffer
- Remove supernatant and resuspend in 20 uL 1X Amp ligase buffer
- Circularization
- Prepare 7X uL Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 60C for 2 hours
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (7x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 56 |
dNTP | 1 mM | 600 pmol | 0.6 | 4.2 |
Betaine | 5 M | 15 umol | 3 | 21 |
10X AmpLigase Buffer | 10X | 1X | 2 | 14 |
Amp Ligase | 5 U/uL | 10 U | 1 | 7 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 4.2 | 29.4 |
nf H2O | 1.2 | 8.4 | ||
Total | 20 | 140 |
Continued tomorrow
Buffers
Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 20 mL |
NaCl | 1.5 M | 0.5 M | 3 | 6.66 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 800 uL |
EDTA | 0.5M | 1 mM | 500 | 40 uL |
nf H2O | NA | NA | NA | 12.5 mL |
Low Salt Buffer
Reagent | Stock | Final | Dilution | Amt in 10 mL |
NaCl | 1.5 M | 0.15 M | 10 | 1 mL |
Tris-HCl | 500 mM | 20 mM | 25 | 400 uL |
EDTA | 0.5M | 1 mM | 500 | 20 uL |
nf H2O | NA | NA | NA | 8.58 mL |