Sequencing for drug resistance identification
A. PCR amplification
Control PCR on gel:
load 3 μl PCR products in 1 % agarose gel electrophoresis with 100 V, 45 min.
B. Purification of PCR products
- Equilibration of column: add 500 µl balance solution BL to an absorption column (CB2), centrifuge at 12,000 rpm for 1 min. After discarding the waste liquor, insert the absorption column (CB2) into the collection tube.
- Estimate the volume of PCR solution, then add 5 times the volume of combination solution and mix completely.
- Load the solution of step 2 to the CB2 column which has been put into a collection tube and stay at ambient temperature for 2 min. Centrifuge 60 s at 12,000 rpm and discard the flow through solution in the collection tube, then reconnect the CB2 column to the collection tube.
- Add 700 µl PW wash buffer to the CB2 column, centrifuge at 12,000 rpm for 60 s, and discard the resulting waste liquor in the collection tube.
- Wash the CB2 column with 500 µl PW, centrifuge at 12,000 rpm for 60 s, and discard the waste liquor resulted in the collection tube.
- Recombine the CB2 column to the same collection tube, centrifuge at 12,000 rpm for 2 min to remove the residual washing buffer as clean as possible. Stay at ambient temperature for several minutes until it dry completely. Otherwise, the residual washing buffer will interfere with the result of the next step.
Note: The Ethanol in washing buffer could affect the following steps of enzyme reaction.
- Put the CB2 collumn into a new centrifuge tube, add 15 µl elution buffer dropwisely to the center region of the absorption membrane. Stay at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min to collect the produced DNA solution.
- Load the PCR product on 1 % agarose gel to check the concentration.
C. Sequencing PCR amplification
Cycles for Sequencing
D. Purification of sequencing PCR products
- Add 2 µl of EDTA (125 mM) and 2 µl of NaAC (3 M, pH=5.2) to the bottom of each tube followed by addition of 50 µl absolute ethanol to each tube. Mix the solution thoroughly and leave them at room temperature for 15 min. After centrifugation 30 min at 12,000 rpm, decant the supernatant carefully.
- Add 250 µl of 70 % ethanol, centrifuge 15 min at 12,000 rpm and decant the supernatant carefully .
- Repeat the step 2 again.
- Add 10 µl Hi-Di formamide to dissolve the sample residue after the residual ethanol evaporate at ambient temperature.
- Denature the purified product at 95 °C and chill the product quickly in an ice bath for 4 min, then load 10 µl of the sample to electrophoresis wells.
- Procedure for sequencer instrument
Pre-warm the instrument and activate the signal collection software (Date Collection v3.0). Follow the instructions on the instrument display and load the sample until the instrument is ready. There should be no bubbles in the 96-well plate. Select the optimum running, analysis scheme and date-saved folder, click on start. After finishing sequencing, activate the Sequencing Analysis software to obtain the electrophoresis spectra and gene sequences.
- Align the sequence