We recommend setting up 10 ml of PCR master mix and aliquoting it into a 96-well plate, 100 µl per well. This protocol is scaled for a starting amount of 10 ml; simply scale the volumes in the later sections accordingly if you start with a different scale of PCR. In our hands, this typically results in a final yield of 500 – 2000 nmole of ssDNA FISH probe.
- 200 µM unlabeled “R” primer stock
- 200 µM labeled “F” primer stock
- 10X PCR buffer (KAPA Buffer A) or equiv.
- 10 mM dNTP mix
- Molecular biology grade ddH2O
- 1 ng/µl complex DNA library (or 0.01 µM library if working with commercial oligos)
- Taq DNA polymerase (e.g. KAPA Taq)
- 10 µl 10x buffer
- 2 µl 10 mM dNTP mix
- 0.5 µl 200 µM “R” unlabeled primer
- 0.5 µl 200 µM “F” labeled primer
- 1 µl 1 ng/µl complex DNA library (or 1 µl 0.01 µM library)
- 1 µl KAPA Taq
- 85 µl ddH2O
- “Touch-up” PCR steps (for stepping up library from 21 bp primers to 53 bp secondary-compatiable primers):
- 95°C 5:00
- 95°C 0:30
- 60°C 0:30
- 72°C 0:15
- Repeat 2x (3 cycles total with annealing T of 60°C)
- Move to the below program
- (do everything at 60C if using original unextended primers approach)
- 95°C 0:30
- 68°C 0:30
- 72°C 0:15
- Repeat 39x (40 cycles total with annealing T of 68°C)
- 72°C 5:00
- End
- Molecular biology grade glycogen, 20 mg/ml (Thermo #R0561)
- 4 M ammonium acetate (Sigma A1542)
- Ice-cold 100% ethanol
- Ice-cold 70% (v/v) ethanol in ddH2O
- Pool the cycled PCR reactions and collect in a 50 ml conical tube
We find the quickest way to do this is to use a multichannel pipette and a reagent reservoir in a PCR or tissue culture hood.
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Add 190 µl of glycogen, 1 ml of 4M ammonium acetate, and 25 ml of ice-cold 100% ethanol. Vortex vigorously
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Transfer to 2 ml eppendorf tubes, 2 ml each (~18 tubes)
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Incubate at -80°C for 35’ or at -20°C for at least 2 hours
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Spin at max speed for 1 hour at 4°C
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Aspirate off the supernatant, taking care not to disturb the pellet
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Add 1350 µl of ice-cold 70% ethanol to each tube
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Spin at max speed for 30 minutes at 4°C
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Aspirate off the supernatant, taking care not to disturb the pellet
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Air dry the pellets for 15’ on a 42°C heat block
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Add 40 µl of ddH2O to each tube
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Incubate at 37°C for 30-60 minutes
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Proceed to the digestion step directly, else store at 4°C
- NEB buffer 2
- Nb.BsrDI (NEB R0648)
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Pool the precipitated PCR products into one eppendorf tube (~720 µl)
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Add 10 µl ddH2O, 90 µl buffer 2, and 80 µl Nb.BsrDI
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Vortex the digestion mix and split into PCR strip-tubes, 25 µl per tube (~36 total)
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Run the following program in a thermocycler: 65°C for 4 hours -> 80°C for 20 minutes -> 4°C
The digestion products can be left at 4°C or concentrated via precipitation immediately
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Pool the digestion reactions (~900 µl), and split 2 x 450 µl into 2 ml eppendorf tubes
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To each, add 10 µl of glycogen, 50 µl of 4M ammonium acetate, and 1350 µl of ice-cold 100% ethanol. Vortex vigorously
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Incubate for 35’ at -80°C or at -20°C for at least 2 hours
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Spin at max speed for 1 hour at 4°C
-
Aspirate off the supernatant, taking care not to disturb the pellet
-
Add 1350 µl of ice-cold 70% ethanol to each tube
-
Spin at max speed for 30 minutes at 4°C
-
Aspirate off the supernatant, taking care not to disturb the pellet
-
Air dry the pellets for 15’ on a 42°C heat block
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Add 60 µl of ddH2O to each tube
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Incubate at 37°C for 30-60 minutes
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Proceed to the electrophoresis step directly, else store at 4°C
Modified to use in-house cast gels and Zhuang Lab equipment. Original recovery was just to percipitate the extract. This protocol adds a 1-Butanol extraction to allow a larger volume of water to dissolve gel and reconcentrate product prior to precipitation.
- 2x Loading Buffer (96% formamide/20mM EDTA, prepare using freshly deionized formamide)
- Acrylamide/Bis 19:1 40% (w/v) Solution
- TEMED
- 10% APS (w/v). make fresh from powder and store frozen aliquots (<3 months)
- 1-Butanol
- Prepare 100 mL of acrylamide solution. For a 12% gel:
- Urea 48 g
- 40% Acrylomide 30 mL
- 10x TBE buffer 10 mL
- ddH2O 20 mL
- When urea is dissolved, add 150µL of TEMED and 300mL of 10% APS. Mix thoroughly, then pour between gel plates.
- Insert comb, and let gel polymerize for > 45 min.
- Remove sealing tape and insert gel into gel running apparatus. Fill with 0.5x TBE.
- Pre-run gel at 50mA for > 1hr.
- Dissolve the precipitated oligos in 30µL TE and add an equal volume of 2x loading buffer.
- Heat samples for 5 min at 95°C.
- Load samples onto gel and run gel at 50mA.
- Cut out band, crush using pestle, add to a 15mL Falcon tube add 4mL of nuclease free H2O (or TE) then incubate overnight at 37°C with shaking. [PAUSE POINT]
- Spin down PAGE extraction, save supernatant, and extract gel fragments with an additional 2 mL of nuclease free H2O (or TE) for >2 hrs.
- Spin down PAGE extraction and combine with previous supernatant.
- Perform butanol extractions
- add 3 volumes of 1-BuOH to the sample,
- vortex to mix,
- spin briefly to separate layers (1000 RPM 30s),
- discard organic [top] layer (butanol-waste).
- Repeat until the volume is < 300 µL.
- If residual polyacrylamide fragments are present, centrifuge aqueous layer to remove gel fragments.
- Ethanol precipitate the DNA:
- add 1/10 volumes of 3M NaOAc, mix,
- add 2.5 volumes (including the NaOAc) ice-cold EtOH.
- Incubate at -20°C for > 30 min, then spin for 30 min at max speed in cold room.
- Pour off supernatant, add 0.5 mL of ice-cold 80% EtOH to pellet, spin for 5 min at room temp, and pour off supernatant.
- Dry pellet by air drying (15’ on a 42°C heat block) or using the speed vac. Do not overdry single-stranded DNA (may degrade).
- Resuspend in 60 µl of ddH2O, incubate at 37°C for 30-120 minutes
- Quantify probe yield by spectrophotometry using a fluorometer or Nanodrop (microarray setting) on the fluorophore (i.e. read out pmoles/µl of fluor).