1 | Prepare intact, high-quality genomic DNA samples each containing 1 µg at a high concentration (at least 250 ng µl-1). Dilute all samples to the same volume (4 µl) with nuclease free water.
2 | Prepare a master mix for restriction digest by combining the following components in a single tube and mixing thoroughly by back-pipetting. The volumes listed here are intended for a single reaction, so multiply by the number of samples plus some small amount for pipetting error.
0.6 µl 10X Buffer R
0.4 µl 150 µM SAM
1.0 U AlfI
Nuclease-free water (NFW) to total volume 2.0 µl
3 | Combine 2 µl master mix with each 4 µl DNA sample, and incubate 1 hr at 37°C, then inactivate the enzyme at 65°C for 20 min and hold samples on ice.
1 | Prepare two partially double stranded adaptors by combining each pair of oligonucleotides at a final concentration of 4 µM each. Adaptor 1 is prepared by combining sld-ada1-AlfI and anti-sld-ada1, and adaptor 2 by combining sld-ada2-AlfI and anti-sld-ada2 (Table 1).
2 | Prepare a master mix for ligation by combining the following components in a single tube. The volumes listed here are intended for a single reaction, so scale up as needed.
0.5 µl 10 mM ATP
2.0 µl 10X T4 ligase buffer
2.5 µl 5 µM Adaptor 1
2.5 µl 5 µM Adaptor 2
11.5 µl NFW
3 | Combine 20 µl master mix with 5 µl of digested DNA. Incubate for 1 hour (16°C for heat-inactivatable enzymes e.g. AlfI, or at 4°C for enzymes that cannot be inactivated, e.g. BsaXI), then hold on ice.
1 | Conduct a test-scale PCR to determine the minimum number of cycles required to produce a visible product, and evaluate relative yield across samples. Prepare a master mix for PCR by combining the following components in a single tube, and mix thoroughly by backpipetting. The volumes listed here are intended for a single reaction, so scale up as needed.
6.5 µl NFW
2.5 µl 2.5 mM dNTP
0.4 µl 10 µM sld-p5
0.4 µl 10 µM sld-p6
1.0 µl 1 µM sld-p3
1.0 µl 1 µM sld-p4 (barcode)
4.0 µl 5X HF buffer
0.2 µl Phusion polymerase
2 | Combine 16 µl master mix with 4 µl ligation from each sample, and amplify on the following profile: (98°C 5 sec, 60°C 20 sec, 72°C 10 sec) X 12 cycles.
3 | Sample 5 µl from each reaction at two-cycle intervals (n = 6, 8, 10, & 12 cycles). The thermocycler can be paused during each sampling interval.
4 | Visualize PCR products using standard gel electrophoresis methods (2% agarose TBE gel), including a low-molecular weight marker. Select the minimum number of cycles required to produce a visible product at ~130 bp.
5 | Prepare a master mix for preparatory-scale PCR by combining the following components in a single tube, and mix thoroughly by backpipetting. The volumes listed here are intended for a single reaction, so scale up as needed.
32.5 µl NFW
12.5 µl 2.5 mM dNTP
2.0 µl 10 µM sld-p5
2.0 µl 10 µM sld-p6
5.0 µl 1 µM sld-p3
5.0 µl 1 µM sld-p4 (barcode)
20.0 µl 5X HF buffer
1.0 µl Phusion polymerase
6 | Combine 80 µl master mix with 20 µl ligation and amplify using the optimal cycle number determined in step 4.
1 | Resolve samples on 2% agarose gels using standard electrophoresis methods and reagents. Large wells are required to accommodate each 100 µl sample in a single well.
2 | Using a UV transilluminator set to low intensity, excise the target band (~130 bp) from each lane, being careful to limit the UV exposure of each sample to no more than 30 seconds.
3 | Extract preparations from excised gel slices either:
A. using a commercial gel-extraction kit of your choice according to the manufacturers instructions; or
B. by eluting directly from the gel slice into water (40 µl NFW in a 1.5 ml microcentrifuge tube). Gel slices are held overnight at 4°C, and the eluent collected the following morning.
4 | Collect a small aliquot (~2-10 µl) from each preparation and combine in a multiplex pool for sequencing. Remaining preparations can be held for 6 months at -20°C and sampled again for sequencing if additional coverage is required for any samples.