222
- With the help of a gel cutter, slice out the band with the
approximately desired band size and transfer it into a micro-
centrifuge tube for PCR cleanup. - The excised gel containing the band of interest is then purified
using a PCR cleanup kit according to the manufacturer’s
protocol. - Briefly, dissolve the excised gel with the solution provided at
56 °C for 10 min, vortexing occasionally to help the dissolving
process. Then transfer the mixture onto the column provided
and spin it at 11,000 × g. Discard flow-through and wash the
column twice with washing buffer (see Note 12). Finally, add
30 μL of DNase-free water onto the membrane of the column
(see Note 13) and elute the purified gel product. - Measure the concentration, A 260 /A 280 ratio, and A 260 /A 230
ratio of the resulting elution using a nanospectrophotometer. - Store the elution in − 20 °C freezer until further use.
- There are many Sanger sequencing services commercially avail-
able, which may prove cost-effective. If performing your own
Sanger sequencing, proceed with the following steps. - Thaw all required ingredients for Sanger sequencing prepara-
tion on ice away from the light. - Briefly tap and spin down each reagent to ensure homogeneity
(see Note 14). - Prepare run sequencing reaction according to the manufactur-
er’s protocol for both forward reaction (with forward primer)
and reverse reaction (with reverse primer) (see Note 15).
IMPORTANT! Change pipette tips after each pipetting to
prevent cross contamination. - Try to prevent formation of bubbles as much as possible (see
Note 16). - Place the sample into the thermal cycler and amplify the sam-
ple to endpoint according to the manufacturer’s protocol (see
Note 17). - Store the samples at 4 °C until ready for purification process.
- Thaw all required ingredients for purifying sequencing reac-
tion on ice (see Note 18). - Briefly tap and spin down each reagent to ensure homogeneity
(see Note 19). - Prepare purifying sequencing reaction mix according to the
manufacturer’s protocol (see Note 20). - Resuspend the dried sample in 10 μL of highly deionized for-
mamide (see Note 21). - Vortex to mix and then centrifuge to bring the mixture back
to the bottom (see Note 22).
3.4 Sanger
Sequencing
Reaction Preparation
3.5 Purifying the
Sequencing Reaction
Katherine T. W. Lee et al.