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• step description "Add an equal volume of isopropanol to tube. Mix contents gently and keep undisturbed for 5-10 min. The white thread-like structure of precipitated DNA will be seen in suspension" assertion.
• step description "Wash DNA pellet by adding 10 ml of 75% ethanol. Centrifuge at 12,000 x g for 10 min, 4 °C." assertion.
• step description "Add 100 µl 3 M sodium acetate solution per ml of aqueous phase obtained in step 10. Mix gently by inverting tube." assertion.
• step description "Centrifuge suspension at 12,000 x g for 10 min, 4 °C. Carefully transfer upper aqueous phase to a new 50 ml tube." assertion.
• step description "Wash (centrifuge in 100 x g for 10 min) twice with 10 ml of sterile PBS or sterile Dulbecco's modified eagle medium. The approximate yield of cells from 4 ml of blood varies between 107-108." assertion.
• step description "Aspirate the whitish buffy coat (about 1 ml) (PBMCs) formed in the interphase between histopaque and medium." assertion.
• step description "Gently layer the blood on the top of Ficoll Histopaque using a 1 ml auto pipette. The layering should be done very slowly that blood and Ficoll Histopaque should stay as two different layers." assertion.
• step description "Collect 4 ml of human venous blood sample in heparinised vials (BD biosciences) and mix well by gently inverting the tube several times." assertion.
• step description "Seed 500 µl of cell suspension in a 24 well culture plate. Note: Monocytes in PBMCs get attached to the plastic in about 2-3 h when incubated at 37 °C. Longer incubation will result in firm attachement. Lymphocytes are not glass adherent and they will be mostly in suspension and can be removed by mildly flushing the wells with medium and/or buffer. Such treatment will keep the monocytes firmly attached to the surface of culture plates." assertion.
• step description "Count cells by haemocytometer using W.B.C. diluting fluid: Add 10 µl of cell suspension to 190 µl of W.B.C. diluting fluid and mix well. Load the cell suspension in a haemocytometer and count the cells. Adjust cell concentration at 1 x 106 cells/ml with Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution and 10% FBS. The approximate yield of cells from 4 ml of blood varies between 107-108." assertion.
• step description "Wash cells (centrifuge at 100 x g for 10 min) with 10 ml of sterile DMEM (without FBS) twice. Note: Cold DMEM is not used routinely for washing lymphocytes from culture cavities while setting up cultures. Rather when monocytes bound tightly to plastic cavities are needed to be harvested pre-chilled DMEM can be used. " assertion.
• step description "Collect about 4 ml of human venous blood sample in heparinised vials and mix well by gently inverting the tubes several times." assertion.
• step description "After incubating at room temperature for a few minutes, keep the solubilized nucleic acid in −20˚C for a short time storage or in −80˚C for a longtime storage. Figure 1 shows a schematic model of all the DNA and RNA isolation steps of this procedure." assertion.
• step description "Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended." assertion.
• step description "Dissolve the pellet in 20-30 µl of RNase free water (commercial) or autoclaved water." assertion.
• step description "Discard the supernatant and wash the precipitate nucleic acid gently with 70% EtOH." assertion.
• step description "Add 700 µl of cold isopropanol to precipitate RNA or DNA and then invert tubes 3-4 times to mix the solution." assertion.
• step description "Centrifuge at 13700 g at 4℃ for 10 min.CRITICAL STEP The PH of the extraction buffer is about 8-9, resulting in DNA and RNA precipitation, however, it could result in lower DNA and higher RNA concentrations in case of reducing the PH to around 6-7." assertion.
• step description "Add 600 µl of chloroform: isoamyl alcohols (24:1) to each tube, homogenize them by vortexing." assertion.
• step description "Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended." assertion.
• step description "Count cells by haemocytometer using W.B.C. diluting fluid: Add 10 µl of cell suspension to 190 µl of W.B.C. diluting fluid and mix well. Load the cell suspension in a haemocytometer and count the cells. Adjust cell concentration at 1 x 106 cells/ml with Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution and 10% FBS. The approximate yield of cells from 4 ml of blood varies between 107-108." assertion.
• step description "Cell lysates were transfer into eppendorf tubes and one-fifth (volume/volume) of chloroform was added to each tube. " assertion.
• step description "Cells were seeded in 100-mm-diameter dishes and infected with either 2 PFU of live IBV per cell or the same amount of UV-inactivated IBV (UV-IBV) at 37 °C. Excess virus in the medium was removed by replacing with fresh medium at 1 h post-infection." assertion.
• step description "RNA concentration and purity were determined by NanoDrop." assertion.
• step description "RNA pellet was washed with 1 ml 70% RNase-free ethanol once and spin down by 7,500 x g for 5 min." assertion.
• step description "Shake tubes vigorously by hand for 15 sec and incubated for 3 min at room temperature, then centrifuged at 12,000 x g for 15 min at 4 °C." assertion.
• step description "At the indicated time points (0, 2, 4, 8, 12, 16, 20, 24, 28 h post-infection), cells were rinsed with 10 ml Phosphate Buffered Saline (PBS) buffer once and lysed in 1 ml TRIzol for 5 min at room temperature." assertion.
• step description "Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended" assertion.
• step description "Add 600 µl of chloroform: isoamyl alcohols (24:1) to each tube, homogenize them by vortexing" assertion.
• step description "Ground samples (leaf, shoot, root, and recalcitrant samples, approximately 0.5-1 g) using 1 ml of the extraction buffer with or without liquid nitrogen in mortar and pestle that are sterilized.CRITICAL STEP The procedure are carried out at room temperature except the centrifugation steps (at 4℃) as well as the time of precipitating of the nucleic acid using the isopropanol (on ice).CRITICAL STEP Severely disrupt the tissues to create the glaze mode of samples" assertion.
• step description "Discard the supernatant and wash the precipitate nucleic acid gently with 70% EtOH." assertion.
• step description "After centrifuging at 5400 g at 4˚C for 5 min, remove the supernatant and then air-dry the pellet." assertion.
• step description "Add 700 µl of cold isopropanol to precipitate RNA or DNA and then invert tubes 3-4 times to mix the solution" assertion.
• step description "Centrifuge at 13700 g at 4℃ for 10 min.CRITICAL STEP The PH of the extraction buffer is about 8-9, resulting in DNA and RNA precipitation, however, it could result in lower DNA and higher RNA concentrations in case of reducing the PH to around 6-7" assertion.
• step description "Dissolve the dry pellet in nuclease free water or TE buffer (Ph 8.0). Store DNA at 4 º celcius or -20 º celcius" assertion.
• step description "Wash preserved mosquitoes in sterile distilled water or phosphate buffer saline(PBS) to remove excess alcohol. Fresh mosquitoes can be ground directly." assertion.
• step description "Wash the pellet with 70% ethanol" assertion.
• step description "Transfer the very clear supernatant to a fresh tube , add two fold volume cold isopropanol and keep it for 1 hour at -20º celcius" assertion.
• step description "Add equal volume of phenol:chloroform(1:1) ,shake the tube well for 5 minutes and centrifuge at 12,000g for 10 minutes at 4º celcius" assertion.
• step description "Lyse for 1 hour 30 minutes at 37º celcius. Gently mix the tube by inverting every 15 minutes" assertion.
• step description "Add 2 volumes of cold 95% ethanol and mix gently by inversion. The tubes are incubated at for 45 min in -20°C freezer. It should not be for more time as some remaining phenolics and resin may also precipitate with DNA." assertion.
• step description "Centrifuge the tube for 10 min at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Place the tube for 30 min at 37°C." assertion.
• step description " The falcon tube was kept into the 65°C incubator or water bath and mix gently by inversion after every 10 min till 45 min. After incubation, place the tube at room temperature for five min to reach to room temperature environment. Centrifuge the 50ml falcon tube for 5 min at 3000×g on room temperature. For next generation sequencing, the greater the genome size, lower is speed of initial centrifugation" assertion.
• step description "Take one gram of frozen finely powdered leaf tissues in a new 50 mL Falcon tube and mixed with the pre-heated extraction buffer (10 ml) for one sample. Breifly, vertex for 30sec to ensure leaf material is fully mixed with buffer. Fine grind is a key to obtaining high DNA quantity with lesser artifacts of resin." assertion.
• step description "Add 1 ml of 10% SDS solution (final concentration, ~1%) and 20 µl of 10 mg/ml Proteinase K solution (final concentration 20 µg/ml). Incubate for 2 to 4 h at 60 °C in a hot water bath" assertion.
• step description "Add 10 ml TE buffer into the mortar and carefully bring mycobacteria in suspension. Transfer this suspension to a 50 ml polypropylene tube" assertion.
• step description "Pellet down bacilli by centrifugation at 2,000 x g for 10 min, room temperature and carefully discard supernatant." assertion.
• step description "Harvest mycobacteria by centrifuging 20-40 ml log phase culture (O.D.600 ≈ 0.8) at 2,000 x g for 10 min, room temperature. Carefully discard culture medium and add 10 ml TE (at room temperature) buffer to pellet. Mix gently with a pipette. Note: If isolating DNA from pathogenic mycobacteria such as M. tuberculosis, step 1 and 2 must be performed in a BSL-3 facility." assertion.
• step description "Wash DNA pellet by adding 10 ml of 75% ethanol. Centrifuge at 12,000 x g for 10 min, 4 °C" assertion.
• step description "Add an equal volume of isopropanol to tube. Mix contents gently and keep undisturbed for 5-10 min. The white thread-like structure of precipitated DNA will be seen in suspension (Figure 1)" assertion.
• step description "Repeat steps 6 to 8 for achieving a higher purity of DNA" assertion.
• step description "Add an equal volume of PCI mixture to the tube and mix contents by gently inverting tube for 5-10 min" assertion.
• step description "Dissolve the dry pellet in nuclease free water or TE buffer (Ph 8.0). Store DNA at 4 º celcius or -20 º celcius ." assertion.
• step description "Grind mosquitoes in 1.5 ml eppendorf tube with micropistle in 50-100 µl 1X STE buffer ( 50mM Nacl,50mM Tris- HCL,100mM EDTA, Ph 8.0) along with 100mM sucrose. Add 1X STE buffer to a total volume of 300-500 µl for a single mosquito and 1 ml for mosquito pool like 4,6,8,10 numbers. Then add 1% SDS ,1% Triton -X , 10 µl/ ml Rnase A (20mg/ml), 20 µl/ ml Proteinase K (20mg/ml) and mix it." assertion.
• step description "Keep the pellet at 37 º celcius for 10 minutes." assertion.
• step description "Centrifuge at 12,000g for 30 minutes at 4º celcius and then remove the supernatant ." assertion.
• step description "Repeat the above step(5) , then add chloroform:isoamyl alcohol(24:1) and centrifuge at 12,000g for 10 minutes at 4º celcius ." assertion.
• step description "Lyse for 1 hour 30 minutes at 37º celcius. Gently mix the tube by inverting every 15 minutes." assertion.
• step description "Add 2 volumes of cold 95% ethanol and mix gently by inversion. The tubes are incubated at for 45 min in -20°C freezer. It should not be for more time as some remaining phenolics and resin may also precipitate with DNA." assertion.
• step description "Centrifuge the tube for 10 min at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Place the tube for 30 min at 37°C." assertion.
• step description "The falcon tube was kept into the 65°C incubator or water bath and mix gently by inversion after every 10 min till 45 min. After incubation, place the tube at room temperature for five min to reach to room temperature environment. Centrifuge the 50ml falcon tube for 5 min at 3000×g on room temperature. For next generation sequencing, the greater the genome size, lower is speed of initial centrifugation" assertion.
• step description "The IBV-infected cells were incubated at 37 °C in 5% CO2." assertion.
• step description "Cells were seeded in 100-mm-diameter dishes and infected with either 2 PFU of live IBV per cell or the same amount of UV-inactivated IBV (UV-IBV) at 37 °C. Excess virus in the medium was removed by replacing with fresh medium at 1 h post-infection." assertion.
• step description "Centrifuge tubes at 13700 g at 4℃ for 10 min. The white pellet will be visible on the bottom of the tubes.CRITICAL STEP Do not disturb bottom phases of the solution when you pipet the upper phase." assertion.
• step description "After centrifuging at 5400 g at 4˚C for 5 min, remove the supernatant and then air-dry the pellet." assertion.
• step description "Dissolve the pellet in 20-30 µl of RNase free water (commercial) or autoclaved water." assertion.
• step description "Transfer the upper aqueous phase to a new tube (size= 1.5 ml)" assertion.
• step description "Centrifuge tubes at 13700 g at 4℃ for 10 min. The white pellet will be visible on the bottom of the tubes.CRITICAL STEP Do not disturb bottom phases of the solution when you pipet the upper phase" assertion.
• step description "After centrifuging at 5400 g at 4˚C for 5 min, remove the supernatant and then air-dry the pellet." assertion.
• step description "Dissolve the pellet in 20-30 µl of RNase free water (commercial) or autoclaved water." assertion.
• step description "Centrifuge the tube for 10 min at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Place the tube for 30 min at 37°C." assertion.
• step description "After RNAse A treatment, add one volume of chloroform: isoamyl alcohol again and mix by inversions for 5 min. The tube is centrifuged for 10 min at 5000 × g at 4°C. Transfer the clear supernatant into new 2ml tube and add half volume of 5 M NaCl to the sample and mix gently by inversions." assertion.
• step description "Cells were seeded in 100-mm-diameter dishes and infected with either 2 PFU of live IBV per cell or the same amount of UV-inactivated IBV (UV-IBV) at 37 °C. Excess virus in the medium was removed by replacing with fresh medium at 1 h post-infection." assertion.
• step description "RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4 °C." assertion.
• step description "RNA pellet was washed with 1 ml 70% RNase-free ethanol once and spin down by 7,500 x g for 5 min." assertion.
• step description "At the indicated time points (0, 2, 4, 8, 12, 16, 20, 24, 28 h post-infection), cells were rinsed with 10 ml Phosphate Buffered Saline (PBS) buffer once and lysed in 1 ml TRIzol for 5 min at room temperature." assertion.
• step description "Dissolve pellet in molecular biology grade water and analyze DNA by agarose gel electrophoresis and spectrophotometer for its purity and integrity." assertion.
• step description "Add 1 ml of 10% SDS solution (final concentration, ~1%) and 20 µl of 10 mg/ml Proteinase K solution (final concentration 20 µg/ml). Incubate for 2 to 4 h at 60 °C in a hot water bath." assertion.
• step description "Add 10 ml TE buffer into the mortar and carefully bring mycobacteria in suspension. Transfer this suspension to a 50 ml polypropylene tube." assertion.
• step description "Snap freeze pellet in liquid N2 and immediately transfer to a mortar. Using a pestle, crush bacteria while repeatedly adding liquid N2 (~20 ml) to it." assertion.
• step description "Pellet down bacilli by centrifugation at 2,000 x g for 10 min, room temperature and carefully discard supernatant." assertion.
• step description "Bring down temperature of suspension to ~30 °C and add 20 µl of 100 mg/ml lysozyme solution (final concentration, 2 mg/ml) to it. Incubate suspension at 37 °C for 6 h to overnight in a water bath. Note: In case of fast-growing mycobacteria such as Mycobacterium smegmatis (M. smegmatis), go directly to step 7. However, if DNA is to be isolated from slow-growing species, follow steps 4-6 which involve lysis of cells by a physical method. Because of enhanced disruption of cells, this would significantly enhance yield." assertion.
• step description "Incubate mycobacterial suspension at 80 °C for 1 h in a water bath. This step will facilitate loosening of the mycobacterial cell wall and will also inactivate pathogenic mycobacteria." assertion.
• step description "Harvest mycobacteria by centrifuging 20-40 ml log phase culture (O.D.600 ≈ 0.8) at 2,000 x g for 10 min, room temperature. Carefully discard culture medium and add 10 ml TE (at room temperature) buffer to pellet. Mix gently with a pipette. Note: If isolating DNA from pathogenic mycobacteria such as M. tuberculosis, step 1 and 2 must be performed in a BSL-3 facility." assertion.
• step description "Aspirate liquid without disturbing pellet. Keep tube open to air dry pellet. Do not over dry pellet, as over dried DNA will be difficult to dissolve." assertion.
• step description "Wash DNA pellet by adding 10 ml of 75% ethanol. Centrifuge at 12,000 x g for 10 min, 4 °C." assertion.
• step description "Centrifuge tube at 12,000 x g for 10 min, 4 °C. Discard liquid carefully." assertion.
• step description "Add an equal volume of isopropanol to tube. Mix contents gently and keep undisturbed for 5-10 min. The white thread-like structure of precipitated DNA will be seen in suspension (Figure 1)." assertion.
• step description "Add 100 µl 3 M sodium acetate solution per ml of aqueous phase obtained in step 10. Mix gently by inverting tube." assertion.
• step description "Repeat steps 6 to 8 for achieving a higher purity of DNA." assertion.
• step description "Centrifuge suspension at 12,000 x g for 10 min, 4 °C. Carefully transfer upper aqueous phase to a new 50 ml tube." assertion.
• step description "Add an equal volume of PCI mixture to the tube and mix contents by gently inverting tube for 5-10 min." assertion.
• step description "Snap freeze pellet in liquid N2 and immediately transfer to a mortar. Using a pestle, crush bacteria while repeatedly adding liquid N2 (~20 ml) to it." assertion.
• step description "Harvest mycobacteria by centrifuging 20-40 ml log phase culture (O.D.600 ≈ 0.8) at 2,000 x g for 10 min, room temperature. Carefully discard culture medium and add 10 ml TE (at room temperature) buffer to pellet. Mix gently with a pipette. Note: If isolating DNA from pathogenic mycobacteria such as M. tuberculosis, step 1 and 2 must be performed in a BSL-3 facility." assertion.
• step description "Pellet down bacilli by centrifugation at 2,000 x g for 10 min, room temperature and carefully discard supernatant." assertion.
• step description "Snap freeze pellet in liquid N2 and immediately transfer to a mortar. Using a pestle, crush bacteria while repeatedly adding liquid N2 (~20 ml) to it." assertion.
• step description "Add 10 ml TE buffer into the mortar and carefully bring mycobacteria in suspension. Transfer this suspension to a 50 ml polypropylene tube." assertion.
• step description "Add an equal volume of isopropanol to tube. Mix contents gently and keep undisturbed for 5-10 min. The white thread-like structure of precipitated DNA will be seen in suspension" assertion.
• step description "Aspirate liquid without disturbing pellet. Keep tube open to air dry pellet. Do not over dry pellet, as over dried DNA will be difficult to dissolve." assertion.
• step description "Centrifuge suspension at 12,000 x g for 10 min, 4 °C. Carefully transfer upper aqueous phase to a new 50 ml tube" assertion.

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