Samples are now ready for hybridization to the Helicos Flow Cell for subsequent sequencing-by-synthesis. DNA concentrations of 150–300 pM are utilized for each Helicos Flow Cell Channel in a 20-ml loading volume.
Placed in thermocycler and run the following conditions: 37°C for 60 min, 70°C for 10 min, maintain at 4°C until ready to proceed to next step.
Add 2 ul Terminal Transferase (20 U/ml) to each tube and mix thoroughly by pipetting up and down 10 times. Then collect contents by centrifugation.
Add 0.3 ml of 500 mM Biotin ddATP to each tube.
Heat denature the DNA at 95°C for 5 min in the thermocycler. Remove immediately and snap cool for a minimum of 2 min by placing in the ice-cooled aluminum block.
Determine the double-stranded DNA concentration using a NanoDrop 1000 or 8000 spectrophotometer. Calculate the pmoles of the ends in the sample using the following formula :Pmol3’termini/ µl = XXng DNA/ µl x (103 pg/ng) x (pmole/660 pg) x (1/average fragment size as determined from gel) x 2(3’ termini/ds DNA molecule)
Estimate the size of sheared product by comparing the middle of the DNA smear to the size standards.
DNA standards of 1000 and 25 bp ladders are included for size comparison.
Determine the approximate concentration of 3’ ends to effectively tail the 3’ ends of the genomic DNA. This requires a determination of average fragment size of sheared DNA obtained by running a 2-µl DNA aliquot on a 4-20% gradient Tris Borate EDTA (TBE) polyacrylamide gel.
Twenty microliters of the oligo control is run on a 4–20% polyacrylamide gel in TBE alongside 100 and 25 bp ladders.
Place the tubes in the thermocycler based on the following conditions: 37°C for 60 min, 70°C for 10 min, maintain at 4°C.
Add 15 ml of Sample Tailing Mix or Control Tailing Mix to each DNA tube. Pipette up and down 10 times to mix it and collect liquid contents by cetrifuge.
Place the sheared DNA and tailing oligo control tubes in a PCR Thermocycler, at 95°C for 5 min to denature the DNA. Place tubes in an aluminum block pre-chilled on an ice slurry for 2 min to prevent reannealing of the denatured single-stranded DNA.
Prepare a separate 200-ul PCR tube with tailing control sample that consists of 0.8 pmoles of DNA sample and 0.2 pmoles of tailing oligo control to monitor efficiency of tailing.
Place the 3.0 pmole sheared DNA sample into a 200-ml PCR tube.
Prepare sample of DNA Tailing Mix (1 reaction consist of: 4 ul 10 Terminal Transferase buffer, 4 ul 2.5 mM CoCl2, 2 ul Terminal Transferase Enzyme (20 U/ml), 3.9 ml Helicos, supplied Poly-A Tailing dATP and 1.1 ml deionized water (dH2O).
Collect the 20 µl volume and transfer to a new 1.5 -ml tube. Repeat the process to remove any remaining DNA on AMPure beads. DNA will now be in 40 µl volume.
Add 20 µl of water to elute the sheared DNA, pipette the beads and water up and down 20 times and place the tube back on Dynal magnet.
Wash beads twice with 700 µl freshly prepared 70% (v/v) ethanol. After that centrifuge, place it on magnet, remove ethanol, and dry pellet completely at RT for 5-7 min. Cracks will form when pellet is dry.
Incubate the tube for 30 minutes with shaking at every 10 minutes. Then, centrifuge at low speed, capture beads on Dynal magnetic stand for 5 min and aspirate supernatant carefully.
Transfer the DNA sample to 1.5 -ml tube and add water until reach 100µl. Vortex and add 300 µl AMPure Bead slurry.
Adjust the DNA volume to 100µ and warm AMPure Bead solution to room temperature (RT). Vortex to resuspend
Next, transfer the DNA into a clean 1.5 -ml microtube. This DNA sample can be stored at -20 ̊ C.
Prepare 1-3 µg of genomic DNA in a final volume of 120 µl 10 mM Tris 1 mM EDTA (1 x TE).
Different fragmentation sizes have different conditions. For example, shearing of DNA to 200 bp required to be done in Covaris microTubes using 3 cycles of 60 s, 10% duty cycle, intensity of 5 and 200 cycles per burst.
For complete coverage, the method chosen must cleave the DNA randomly and provide a 3’ hydroxyl end for subsequent tailing. Ultrasonic shearing of DNA is achieved using the Covaris S2 instrument, resulting in fragmentation that suit for sequencing of entire genomic sample. Covaris allow the use of genomic DNA ranging in length from 100 to 3000 base pairs (bp). For typical genomic DNA sequencing, average size of 200-300 bp for DNA is fragmented. For paired read sequencing in which two or more regions of same DNA fragment sequenced, 1500 bp of fragment is optimal thus provide spacer gaps from 100 to 700 nucleotides in length.