John M. S. Bartlett.pdf - Bio-Nica.info

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50-mL conical tubes. Resuspend in a total of 250 µL of LoTE. (see Note 10 for optional
DNA quantitation at this stage.)
3.10. Isolation of 102-bp Ditags by Gel Purification
1. Load pooled PCR products on three 12% polyacrylamide gels. Run and stain as before.
2. Cut out the 102-bp band of amplified ditags.
3. Fragment gel by placing cut-out bands in 0.5 mL of microcentrifuge tubes that have previously
been pierced through the base with a needle, and insert into a 2-mL tube. (Depending
on bandwidth, 3–4 tubes are used per gel.) Centrifuge at full speed for 2 min.
4. Elute DNA from gel fragments by adding 250 µL of LoTE and 50 µL of 10 M ammonium
acetate to each 2-ml tube. Ensure that gel fragments are covered by buffer (add more if
necessary). Vortex tubes then incubate at 65°C for 2 h.
5. For each 0.5-mL tube, prepare two Spin-X filter microcentrifuge tubes by placing 5 µL of
LoTE on the filter. Transfer contents of each 0.5-mL tube to two Spin-X tubes. Centrifuge
at full speed for 5 min. (see Note 7 and ref. 12).
6. Pool samples and ethanol precipitate. Resuspend in a total of 100 µL of LoTE.
3.11. Isolation of 26-bp Ditags by NlaIII Digestion and Removal
of Linkers Using Magnetic Beads and Gel Purification
1. To the pooled PCR products, add 58 µL of LoTE, 20 µL of 10× NEBuffer 4, 2 µL of 100×
BSA and 20 µL of NlaIII. Incubate at 37°C for 1 h.
2. During NlaIII digestion, add 100 µL of streptavidin Dynabeads to each of two 1.5-mL
tubes. Immobilize beads with magnet and remove supernatant. Wash beads with 200 µL of
1× B+W buffer. Remove buffer when NlaIII digestion is complete.
3. To each of the two tubes containing streptavidin Dynabeads, add 100 µL of 2× B+W buffer
and 100 µL (half) of the NlaIII digestion (11).
4. Incubate with gentle mixing for 15 min at room temp.
5. Immobilize beads with a magnet. Collect supernatant.
6. Wash beads once with 200 µL of 1× B+W buffer and once with 200 µL of LoTE. Collect
supernatant in each case.
7. Pool supernatants and keep on ice to prevent ditag denaturation (13). Discard beads.
8. P/C extract at 4°C. Ethanol precipitate with centrifugation at 4°C (13). Resuspend in 15 µL
of TE (not LoTE).
9. Load on two lanes of a 12% polyacrylamide gel with a 20-bp ladder. Run at 160 V for 2 h
until the bromophenol blue dye front is 3 cm from the bottom. Stain. (Purified ditags
run at 22–26 bp. Released linkers run at 40 bp. Bands between 60–100 bp result from
incomplete NlaIII digestion.)
10. Cut out ditag band running at 22–26 bp.
11. Elute DNA from gel fragments as before, but incubate at 37°C, not 65°C.
12. Ethanol precipitate with centrifugation at 4°C (13). Resuspend in 6.4 µL of LoTE.
3.12. Ligation of Sticky-Ended 26-bp Ditags to Form Concatemers
Then Gel Purification of Concatemers
1. To the purified ditags, add 0.8 µL of 10× ligase buffer and 0.8 µL of T4 DNA Ligase.
Incubate at 16°C for 2 h (or longer, e.g., overnight, if desired).
2. Add loading buffer directly to concatemer ligation. Heat at 65°C for 15 min then chill on
ice for 5 min (14). Load on 8% polyacrylamide gel in one lane with a 100-bp ladder. Run at
130 V for 3 h, until the bromophenol blue is 3 cm from the bottom. Stain.
3. Cut out DNA smear over 500 bp in size.
4. Elute concatemer DNA from gel fragments as before but incubate at 65°C.
5. Ethanol precipitate and resuspend in 6 µL of LoTE.