1. Take the appropriate quantity of DNA to be sequenced (purified and quantified)

• Reaction mixture for the PCR in the 200 µL PCR tubes
  • PCR mix
  • PCR (<500bp) Plasmid/PCR (>500bp) Control
  • 2.5X TRR mix 0.5 µL 1.0 µL 1.0 µL
  • 5X Buffer 1.75 µL 1.5 µL 1.5 µL
  • Template as per required as per required 1 µL
  • Primer (2-5p mol) 0.5 µL 0.5 µL 2.0 µL
  • Water - µL - µL 4.5 µL
  • Total 10 µL 10 µL 10 µL
• Vortex the PCR tubes briefly and give a short spin
• Keep for the PCR with fallowing program
  • 96 ° C for 1 min
  • 25 cycles (with Ramp rate 1 ° C/sec)
  • 96 ° C for 10 sec
  • 50 ° C for 5 sec
  • 60 ° C for 4 min
  • Final hold at 4 ° C
  • Big dye terminator v 3.1 Cleanup (Qiagen kit)
• After the PCR collect the PCR tubes and give a short spin
• Take the spin columns (with solution) provided in the sequencing kit
• Loosen the cap to a quarter turn
• Then snap off the bottom
• Keep the column in the collection tube
• Centrifuge at 750 g for 3 min
• Transfer the column to a clean 1.5 ml eppendorff tube (matrix bed)
• Pour the PCR product at the centre of the matrix bed without the tip touching it
• Centrifuge for 3 min at 750 g
• Discard the column and Speed Vac dry the collected sample (10-15 min)
• Resuspend the pellet in 20 µL HiDi
• Vortex and give a short spin
• Transfer the content to plate
• Denature the DNA at 95 ° C for 4 min (using PCR machine)
• Snap chill on ice
• Vortex and short spin
• Place the septa onto the plate and place the PCR plate along with its base and the retainer on to the auto-sampler.

Some pints to be remembered:

• Sequencing primers should be dissolved in low TE buffer [Tris (1mM): EDTA (0.01mM)] and the working primers (10 p mol) can be dissolved in autoclaved dd H₂O.
• When 10p mol is working primer conc. If we take 0.5 µL then final conc in the PCR mix would be 5 p mol which is ideal for sequencing

                2.5X + 5Y = 1 x 10, If X =1, 2.5 + 5Y = 10, 5Y = 10 – 2.5 Y = 7.5/5 = 1.5

• HiDi formamide - 10 µL dissolution is ok [never freeze though repeatedly as it forms formic acid]. Make aliquots of 100 µL and keep in deep freezer finish each aliquots in one or two sequences,
• Keep the polymer at 4 degrees for 1 month
• Before filling the polymer, keep the polymer at room temperature for 30 to 45 min and then use
• Before using polymer, 1-2 times conditioning buffer wash
• RFID- Radio frequency Identification
• StdSeq50_POP7 – is to be set for sequencing

Big dye terminator v 3.1 Cleanup (Plate Method)

• Make stocks of
  • 100% Ethanol
  • 0.5M EDTA (pH 8.0) – and freshly prepare 125mM EDTA every week from 0.5M EDTA
  • 3M NaOAc (pH 4.6)
  • 70% Ethanol (use the Milli-Q water for making 70%)
• Milli Q Water
  • Make the master mix I (MM-I) of 10 µL Milli-Q water per reaction 2 µL 125mM EDTA
  • Make the master mix II (MM-II) of 2 µL of 3M NAOAc per reaction 50 µL ethanol (100%)
  • Add 12 µL MM-I to each reaction containing 10 µL of reaction and ensure that the contents are mixed
  • Add 52 µL of MM-II to each reaction
  • Seal the plate with adhesive cover and ensure the contents are mixed by inverting the plate few times
  • Incubate at room temperature for 15 min
  • Spin at a speed of 3000g for 30 min at room temperature
  • Decant the supernatant by inverting the plate on the paper towel (do not tap the plate)
  • Spin the inverted plate upto 180g (once the centrifuge comes to 180g stop the centrifuge) to remove the residual supernatant (remove the wrap cover before spinning)
  • Add 100 µL of 70% ethanol, seal the plate and spin at 3000g for 5 min at room temperature
  • Repeat the 70% ethanol wash step one more time (step 8-10)
  • Add 10 µL HiDi formamide (pipette up and down for 4-5 times for mixing), short spin the plate, denature (95° C for 5 min), snapchill (4° C for 5 min)
  • Cover the plate with the new septa
  • Plate is ready for sequencing

Big Dye Terminator v 3.1 Cleanup (Tube method)

• Make stocks of
  • 100% Ethanol
  • 0.5M EDTA (pH 8.0) – and freshly prepare 125mM EDTA every week from 0.5M EDTA
  • 3M NaOAc (pH 4.6)
  • 70% Ethanol (use the Milli-Q water for making 70%)
• Milli Q Water
  • Make the master mix I (MM-I) of

    10 µL Milli-Q water per reaction

    2 µL 125mM EDTA

  • Make the master mix II (MM-II) of

    2 µL of 3M NAOAc per reaction

    50 µL ethanol (100%)

  • Add 12 µL MM-I to each reaction in the tube containing 10 µL of reaction mix and ensure that the contents are mixed
  • Add 52 µL of MM-II to each reaction tube
  • Mix well the contents and incubate at room temperature for 15 min
  • Spin at a speed of 12000g for 20 min at room temperature
  • Decant the supernatant and keep the tubes inverted on a tissue paper
  • Add 250 µL of 70% ethanol and spin at 12000g for 10 min at room temperature
  • Decant the supernatant and VacDry the tubes
  • Add 10 µL of HiDi to each the tubes, mix by pipetting up and down
  • Transfer the samples to the plates and cover with septa, denature (95° C for 5 min), snap chill (4° C for 5 min) and proceed for the sequencing