Daniel:Notebook/ComboLock/2017-3-8
Padlock0601 Test (Started Wed March 1)
Bglii Digest (Sense)
From the results last week, I realized that the original primer for Bglii cutting (the DNA must be double stranded and the RCA product is single stranded) was in the wrong sense. This is because the RCA product will be the reverse complement of the circular product. So I'm repeating the Bglii digest from last Thursday using the correct primer.
Also, because I'm having trouble keeping track of everything, here are all the letter codes used for the experiment. For future readers, just pay attention to the conditions and maybe sample numbers (Sample 1: Sample, 2: No Template, 3: No C Probe, 4: No PO4 on padlock).
Sample Matrix
Letter Code | Condition | Samples |
X | PostRCA-Unpurified | 1-4 |
Y | PostRCA-Purified | 1-4 |
Z | Unpurified post Bglii Digest (X) | 1, 4 |
Q | Purified post Bglii Digest (Y) | 1, 4 |
S | MaxRCA-Unpurified | 1-4 |
T | MaxRCA-Purified | 1-4 |
V | Unpurified post Bglii Digest (S) | 1, 4 |
W | Purified post Bglii Digest (T) | 1, 4 |
J | Unpurified Bglii Digest-Sense (X) | 1-4 |
K | Purified Bglii Digest-Sense (Y) | 1-4 |
- BglII Digestion
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following 16.2X master mix
- Aliquot 14 uL master mix into new tubes
- Add 5 uL appropriate sample to each tube
- Use the following thermocycler program
- 5 min 95C
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- qPCR
- Make 17.2X qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 430 uL SYBR Fast
- 17.2 uL 10 uM AmpF
- 17.2 uL 10 uM AmpR-Ind21
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170308-Padlock0601-BgliiSense.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
- TBE Gel
- Mix 176 uL TBE, 44 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- 20170308-qPCR-Padlock0601-Bglii-Sense.png
qPCR curves
- 2017-03-08-Padlock0601-BgliiSense-Unpurified.png
Gel image - unpurified samples
- 2017-03-08-Padlock0601-BgliiSense-Purified.png
Gel image - purified samples
- 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 following 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
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Repeat wash step above
- Lock and Latch Hybridization/Ligation
- Set up the following master mix
- Add 19 uL probe mix to each sample
- Incubate at 55C for 10 minutes
- Incubate at 25C for 10 minutes
- Lower the temperature to 20C; wait 30 seconds then take off incubator
- Add 1 uL T4 Ligase to each reaction
- Incubate at RT for 15 min
- Heat kill enzyme by incubating at 65C for 10 minutes Continued tomorrow
Reagent | Single Rxn Vol (uL) | 16.2X MM uL |
RCA Reaction Product | 5 | 0 |
Bglii Cutter Primer (10 uM) | 2 | 32.4 |
10X Buffer 3.1 | 2 | 32.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 162 |
Total | 20 | 226.8 |
Results
Bolt Test
It actually means "Lock Test", but I prefer the "bolt" terminology. Anyway, this experiment is to test for the existence of the connected C probe (with the bolt in the middle) product. This will give me an idea of the efficiency of the reaction as well as the effectiveness.
Protocol
Sample Matrix
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No C probes |
Sample 4 (AB) | No phosphate on PCCB (Mirror C v2) |
Reagent | uL per Sample | Exp uL Total | No C uL Total | NP uL Total |
PCCA-03 (10 uM) | 1 | 4 | 0 | 2 |
PCCB-03 (10 uM) | 1 | 4 | 0 | 0 |
Mirror BC-v2 | 1 | 0 | 0 | 2 |
Wash buffer | 3 | 12 | 10 | 6 |
Total | 5 | 20 | 10 | 10 |
Protocol-Part 2
Reagent | Stock Conc | Final Conc./Amt | uL added | Master Mix (8.2X) |
T4 Ligase Reaction Buffer | 5X | 1X | 4 | 32.8 |
Latch Oligo | 10 uM | 10 pmol | 1 | 8.2 |
Lock Oligo | 10 uM | 10 pmol | 1 | 8.2 |
nfH2O | NA | NA | 13 | 106.6 |
Total | 19 | 155.8 |