Daniel:Notebook/ComboLock/2016-8-17
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Positive Control Amplicon Production (Started yesterday)
Protocol
- Size select gel
- Mix 30 uL TBE, 6 uL loading dye, and 4 uL sample for C1-C2 and C3-C2
- Mix 15 uL TBE, 3 uL loading dye, and 2 uL ladder
- Add 20 uL to each lane (2 lanes for C1-C2 and C3-C2)
- Run the gel at 250V for 25 min
- Stain with 3 uL SYBR gold for 3 min
- Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube
- 2016-08-17-PosControlAmplicon-SizeSelect.png
Before Image
- 2016-08-17-PosControlAmplicon-SizeSelect-After.png
After Image
- Shred the gel by centrifuging at 14000rpm for 1 min 30 sec
- Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking
- Centrifuge at 12000rpm for 1.5 min
- Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm
- Ethanol Precipitation
- Move flow through to a 2 mL tube
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue
- Incubate at -80C for 4 hours
- Pellet by centrifuging for 25 min at 12000rpm at 4C
- Remove supernatant and add 750 uL chilled 70% EtOH
- Centrifuge for 12 min at 12000 rpm and 4 C
- Remove supernatant and air dry
- Resuspend in 30 uL nfH2O and measure in nanodrop
- Latch and Padlock Hybridization
- Mix together samples according to table; use 100 uL LPH buffer with 100 nM Padlock0001-68
- Incubate for 1 hr at 37C
- Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
- Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
- For sample 3, use 10 uL 100 uM Latch0001 (10 uM final concentration)
- Incubate for 30 min at 37C
- Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
- Circularization
- Prepare 20 uL KLN mix
- Prepare 4.1x master mix of Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
- Add 2 uL KLN mix to 20 uL of reaction for sample 1
- Incubate at 55C overnight (XX hours)
- Continued tomorrow
Nanodrop Results
Positive Control Amplicon Test
Sample No | Sample Name | LatchX2 | LatchX3 | PCAmp1 | PCAmp3 | Expected Product |
1 | Amp1 Test | X | X | Amp1 | ||
2 | Amp3 Test | X | X | Amp3 | ||
3 | LatchX2 Specificity | X | X | X | Amp1 | |
4 | LatchX3 Specificity | X | X | X | Amp3 | |
5 | No Template | X | X | None | ||
6 | No Latch | X | X | None | ||
7 | No Padlock | X | X | X | X | None |