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

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4. Load all of the samples onto the automated DNA sequencer fitted with a 6% polyacrylamide
gel using the manufacturer’s software.
3.5. Preparation of 6% Polyacrylamide Sequencing Gel (see Note 3)
1. Place 40 g of urea, 12 mL of 40% acrylamide stock, 20 mL of dH 2 O, and 1 g of mixedbed
ion-exchange resin into a beaker and stir gently while warming. Continue to stir the
solution until all the urea crystals have dissolved (see Notes 8 and 9).
2. Filter the acrylamide through a 0.2-µM filter, degas for 5 min, and transfer to a 100-mL
cylinder.
3. Add 8 mL of filtered 10× TBE buffer and adjust the volume to 80 mL with dH 2 O.
4. To polymerize the gel, add 400 µL of 10% APS (freshly made) and 45 µL of TEMED.
Gently swirl to avoid adding air bubbles.
5. According to the instructions provided for the automated DNA sequencer, run the
sequencing gel and analyze the readouts as indicated in Fig. 1 (see Note 10).
4. Notes
1. Both purification steps can also be performed by spin columns, such as Centri-Sep or
Quick Spin. Although they provide a quicker purification, they are an expensive alternative
for those on a tight budget.
2. The PCR primers can be used as the DNA sequencing primers, and should be at least an
18 mer in length. Increasing the length will increase specificity and prevents priming at a
secondary site. It will also decrease the chances for nonexact hybridization.
3. The GC content of the primer should be between 50 and 60%.
4. After ethanol or isopropanol precipitation, it is very important that the supernatant be
carefully aspirated because the pellets are unstable and might be lost.
5. The dried sequencing pellet can be stored in the dark at 4°C for several days if required.
However, once the loading buffer has been added, the samples should be loaded within
a few hours.
6. The sequencing gel must polymerize for at least 1 h before use. A good time to prepare
the gel is during the PCR of the cycle sequencing reactions.
7. Although there are various ready-made acrylamide solutions on the market, it is recommended
that you make your own solutions because better resolution will be achieved.
However, 40% acrylamide can be purchased ready-made and gives good results.
8. It has been shown that the use of formamide in the polyacrylamide gels can resolve
compressions (13).
9. During the phenol:chloroform extraction, if after the first spin two separate layers are not
seen, then revortex the samples for 1 min and recentrifuge, after which an aqueous and
organic phase should be obtainable.
10. If using the ABI 373A “Stretch” automated DNA sequencers, then it is better to use a
4.75% polyacrylamide gel, and from these machines, it is possible to obtain up to 1 kb
of sequence with one run.
11. If the thermal cycler in your laboratory is not a Perkin–Elmer Cetus, then it will be
necessary to optimize your thermal cycler for the sequencing reactions.
12. The addition of blue dextran to the loading buffer will make gel loading easier.