Part 1 – PCR amplification

Notes:

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.

Reagents:

• 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)

PCR Master Mix:

For 10 mL (96 well plates 100uL).

• 1 ml 10x buffer
• 200 µl 10 mM dNTP mix
• 50 µl 200 µM “R” unlabeled primer
• 50 µl 200 µM “F” labeled primer
• 1o ng emPCR product template
• 100 µl KAPA Taq
• 8.500 ml ddH2O

PCR Program

• “Touch-up” PCR steps (for stepping up library from 21 bp primers to 53 bp secondary-compatiable primers):

1. 95°C 5:00
2. 95°C 0:30
3. 60°C 0:30
4. 72°C 0:15
5. 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)

1. 95°C 0:30
2. 68°C 0:30
3. 72°C 0:20
4. Repeat 39x (40 cycles total with annealing T of 68°C)
5. 72°C 5:00
6. End

Part 2 – DNA precipitation of PCR products

Reagents:

• 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

Procedure:

1. 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.

1. Add 190 µl of glycogen, 1 ml of 4M ammonium acetate, and 25 ml of ice-cold 100% ethanol. Vortex vigorously

2. Transfer to 2 ml eppendorf tubes, 2 ml each (~18 tubes)

3. Incubate at -80°C for 35’ or at -20°C for at least 2 hours

4. Spin at max speed for 1 hour at 4°C

5. Aspirate off the supernatant, taking care not to disturb the pellet

6. Add 1350 µl of ice-cold 70% ethanol to each tube

7. Spin at max speed for 30 minutes at 4°C

8. Aspirate off the supernatant, taking care not to disturb the pellet

9. Air dry the pellets for 15’ on a 42°C heat block

10. Add 40 µl of ddH2O to each tube

11. Incubate at 37°C for 30-60 minutes

12. Proceed to the digestion step directly, else store at 4°C

Part 3 – Nicking Endonuclease Digestion and Concentration

Reagents:

• NEB buffer 2
• Nb.BsrDI (NEB R0648)

Procedure:

1. Pool the precipitated PCR products into one eppendorf tube (~720 µl)

2. Add 10 µl ddH2O, 90 µl buffer 2, and 80 µl Nb.BsrDI

3. Vortex the digestion mix and split into PCR strip-tubes, 25 µl per tube (~36 total)

4. 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

1. Pool the digestion reactions (~900 µl), and split 2 x 450 µl into 2 ml eppendorf tubes

2. To each, add 10 µl of glycogen, 50 µl of 4M ammonium acetate, and 1350 µl of ice-cold 100% ethanol. Vortex vigorously

3. Incubate for 35’ at -80°C or at -20°C for at least 2 hours

4. Spin at max speed for 1 hour at 4°C

5. Aspirate off the supernatant, taking care not to disturb the pellet

6. Add 1350 µl of ice-cold 70% ethanol to each tube

7. Spin at max speed for 30 minutes at 4°C

8. Aspirate off the supernatant, taking care not to disturb the pellet

9. Air dry the pellets for 15’ on a 42°C heat block

10. Add 60 µl of ddH2O to each tube

11. Incubate at 37°C for 30-60 minutes

12. Proceed to the electrophoresis step directly, else store at 4°C

Part 4 – Electrophoresis, Gel Extraction, and Recovery

Reagents:

• 2X TBE-Urea sample buffer (Bio-Rad 161-0768)
• Low Range DNA ladder (Thermo #SM1211) Ultra Low Range DNA ladder (Thermo #SM1203)
• 15% TBE-Urea polyacrylamide gel (Bio-Rad 345-0092)
• 0.4 M ammonium acetate

Procedure:

We have had success with the Bio-Rad Criterion cell and gels, but any equivalent set- up should work

1. Microwave 1L of 1X TBE buffer for 3.5 minutes on ‘high.’ Repeat 1 additional time

The TBE should be hot but not boiling. We microwave in a glass 1 L bottle, which will be too hot to touch with bare skin but can be handled carefully with a latex or nitrile glove. If the buffer is too hot, the plastic gel casing will crack.

1. Add an empty gel to the gel box and fill with heated buffer. Blast the urea out of the sample wells with a P1000 or syringe. Run the gel (pre-loading) at a constant 15W while the samples are being prepared (15-30 mins, stop after 30 mins if the sample prep is taking longer)

2. Pool the precipitated digestion products (~120 µl) and add an equivalent volume of 2X TBE-Urea sample buffer (120 µl) and vortex

3. Split into 40 µl aliquots in PCR strip tubes

4. Add 10 µl of each DNA ladder to separate PCR strip tubes. To each, add 10 µl of 2X TBE-Urea sample buffer and mix

5. Denature the digest products and ladders at 95°C for 5 minutes in a thermocycler, then transfer directly to ice

6. Pipette the samples and ladders into the gel wells

Note: If you’d like to image the gel on a gel box/scanner before excising the labeled 53 base fragment, we recommend saving a small aliquot of the precipitated digestion and running it on a separate gel

1. Run the gel for ~25-35 minutes

We typically run the gel such that the orange G marker is at the very bottom of the gel (OK to run it off). In our set up, the labeled 53 bp band co-migrates with the xylene cyanol FF band.

1. Remove the gel from its plastic casing and stain with ethidium bromide for 5 minutes

2. Remove the ethidium bromide stain and de-stain with ddH2O for 5 mintues

3. Visualize the gel on a UV box. Cut out the 53 bp band using a razor blade

4. Add each gel slice to a 2 ml eppendorf containing 600 µl of 0.4 M ammonium acetate

5. Incubate overnight at 55°C with shaking

The more shaking the better – a heated vortexer (e.g. Eppendorf Thermomixer) is best, but a shaking incubator such as those used for bacteria and yeast culture is also sufficient

1. The next day, draw up the fluid from each tube using a pipette and transfer to a fresh 2 ml eppendorf

2. Add 13.5 µl of glycogen and 1350 µl of ice-cold 100% ethanol to each tube and vortex vigorously

3. Incubate for 35’ at -80°C or at -20°C for at least 2 hours

4. Spin at max speed for 1 hour at 4°C

5. Aspirate off the supernatant, taking care not to disturb the pellet

6. Add 1350 µl of ice-cold 70% ethanol to each tube

7. Spin at max speed for 30 minutes at 4°C

8. Aspirate off the supernatant, taking care not to disturb the pellet

9. Air dry the pellets for 15’ on a 42°C heat block

10. Add 60 µl of ddH2O to each tube

11. Incubate at 37°C for 30-120 minutes

12. Quantify probe yield by spectrophotometry using a fluorometer or Nanodrop (microarray setting) on the fluorophore (i.e. read out pmoles/µl of fluor while