30 cycles. Annealing 57ºC

Transcription

1 Bioingentech Genomic DNA Purification Rev. Date: February DNA pathogens detection PCR cycling parameters used TIME cycle Initial Denaturation ºC min 0 cycles Denaturation ºC 0 sec Annealing ºC 0 sec Extension ºC 0 sec cycle Final extension ºC min Note: Spin down the tubes of the Kits in a micro centrifuge before opening. Premixes and reagents will be homogenized properly before use. Nuclei lyses solution must be heated to 0ºC for min before use. (DNA purification)

2 DNA Extraction from Shrimp Samples: Hepatopancreas, Gills and Pleopods. Cut the samples and fill the tube until approximately 0 ul mark in a 00 ul tube. Macerate the sample with scissor. Add ml of CLS to wash the sample. Centrifuge to.000 RPM for minutes then discard supernatant. Add 0-00 ul of NLS. Add, - ul of Proteinase K then Vortex times. ul de RNase, Incube to 0-0 C for 0 min. Spin for seconds to.000 RPM. Add 0uL of PPS (Protein Precipitation solution). Vortex times for seconds each. Centrifuge for min to.000 RPM. Take 0uL of supernatant and add to a new tube with. 0uL of Isopropanol. See the white ring in the top of solution. There are the DNA. Homogenize manually two times. Centrifuge for min to.000 RPM. Discard supernatant but keep approximately 0-0uL in the tube bottom. Add 0uL of Wash Solution (remember add ml (for reactions) or ml (for reactions) of Ethanol 0% depending which format you purchased. Homogenize manually three times. Centrifuge for min to.000 RPM. Discard all supernatant. Resuspend the pellet wit -0uL of DNA REHYDRATION SOLUTION (Template).

3 DNA extraction protocol from Birds Take 0uL of bird total blood. Add 0uL of CLS (cell lysis solution). Mix manually times. Incube for min. Centrifuge for min to.000 RPM. Discard supernatant and conserve the red and white pellet. Add above pellet 0uL of NLS (Nuclei lysis solution). Add ul of Proteinase K. Mix by pipetting times using a ml filter tip. ul de RNase, Incube to 0-0 C for 0 min. Spin for seconds to.000 RPM. Add 0uL of PPS (Protein Precipitation solution). Vortex times for seconds each. Centrifuge for min to.000 RPM. Take 0uL of supernatant and add to a new tube with 0uL of Isopropanol. See the white ring in the top of solution. There are the DNA. Homogenize manually two times Centrifuge for min to.000 RPM Discard supernatant but keep approximately 0-0uL in the tube bottom. Add 0uL of Wash Solution (remember add ml (for reactions) or ml (for reactions) of Ethanol 0% depending which format you purchased. Homogenize manually three times Centrifuge for min to.000 RPM Discard all supernatant. Resuspend the pellet wit 0-0uL of DNA REHYDRATION SOLUTION (Template) Then for PCR assay you must take -ul of template and add in ul (,ul of Premix PCR (Remember homogenize your Premix PCR manually three times] + ul of PCR grade water) Mix well by pipetting, add one drop of Mineral Oil and put tubes in thermocycler. Use program mentioned in quality control certificate.

4 DNA extraction protocol from Blood Take 00uL of Blood and add 00uL of CLS then mix manually. Centrifuge minutes to.000 RPM. Eliminate 00uL of supernatant. Add 00uL of CLS. Centrifuge minutes to.000 RPM. Eliminate 0 ul of supernatant. Add above pellet 0uL of NLS (Nuclei lysis solution). Add ul of Proteinase K. Mix by pipetting times using a ml filter tip. ul de RNase, Incube to 0-0 C for 0 min. Spin for seconds to.000 RPM. Add 0uL of PPS (Protein Precipitation solution). Vortex times for seconds each. Centrifuge for min to.000 RPM. Take 0uL of supernatant and add to a new tube with 0uL of Isopropanol. See the white ring in the top of solution. There are the DNA. Homogenize manually two times. Centrifuge for min to.000 RPM. Discard supernatant but keep approximately 0-0uL in the tube bottom. Add 0uL of Wash Solution (remember add ml (for reactions) or ml (for reactions) of Ethanol 0% depending which format you purchased. Homogenize manually three times. Centrifuge for min to.000 RPM. Discard all supernatant. Resuspend the pellet wit 0 ul of DNA REHYDRATION SOLUTION (Template). Then for PCR assay you must take -ul of template and add in ul (,ul of Premix PCR (Remember homogenize your Premix PCR manually three times] + ul of PCR grade water) Mix well by pipetting, add one drop of Mineral Oil and put tubes in thermocycler. Use program mentioned in quality control certificate.

5 DNA extraction protocol from Egg Yolk Put ml of egg yolk in a ml tube. Agrege ml of PBSX and then vortexing times during sec. Centrifuge at 000 rpm for min at Room Temperature. Remove the supernatant and leave about ml in the bottom of tube. Take. ml and pass to ml new tube. Centrifuge for min at 000 rpm then remove the supernatant and leave 0 ul in the bottom of tube. Add 00 ul of NLS and Homogenize by pipeting. Add ul Proteinase K to each tube. Perform Vortex cycles for seconds. Add ul RNAse and incubate 0min in thermoregulated bath between 0 and C. Dry thoroughly each tube and perform Spin Down to retrieve drops of water evaporated. Remove the torula fragment of each tube Add ul of Protein Precipitation solution. Perform Vortex cycles of seconds. Centrifuge at.000rpm for min. Take 00uL supernatant and move to new tube containing 00uL of Isopropanol (grade PA - UPS / BIOTECHNOLOGICAL / MOLECULAR / NO USE TECHNICAL GRADE BIOLOGY), discard the remainder. In new tube Observe DNA ring on top of the solution. Homogenize manually times gently (remember tightly covered tubes) and centrifuged for min.000rpm. Remove supernatant and left approximately ul - 0uL in the bottom of the tube. Add 0uL of 0% ethanol to each tube. (Grade PA - UPS / BIOTECHNOLOGICAL / MOLECULAR BIOLOGY / NO USE TECHNICAL GRADE). Centrifuge for min at.000rpm. Remove all supernatant. Drie all tubes in thermoplate between 0-0 C for minute. When there is no ethanol in the tubes, resuspend the pellet in 0-0uL of DNA Rehydration solution. Add ul of PCR Mix to PCR tubes for each pathogen. Add ul of Template to each tube with PCR Mix. Add to each tube a drop of mineral oil. Run PCR program.

6 DNA extraction protocol from milk Put ml of raw milk in ml tube. Centrifuge 000 rpm for min. Remove fat layer and remaining supernatant. Leave in the bottom ml. Resuspend the ml remaining in the same ml tube. Then take. ml and place in a ml new tube. Centrifuge at,000 rpm for min and then remove the supernatant leaving only about 0 ul in the bottom. Add 0,mL of nucleolysis solution to each tube. Add ul Proteinase K to each tube. Perform Vortex cycles for seconds. Add ul RNAse and incubate 0 min in thermoregulated bath between 0 and C. Dry thoroughly each tube and perform Spin Down to retrieve drops of water evaporated. Add 0uL of Protein Precipitation solution. Perform Vortex cycles of seconds. Centrifuge at.000rpm for min. Take 00uL supernatant and move to new tube containing 00uL of Isopropanol (grade PA - UPS / BIOTECHNOLOGICAL / MOLECULAR / NO USE TECHNICAL GRADE BIOLOGY), discard the remainder. In new tube Observe DNA ring on top of the solution. Homogenize manually times gently (remember tightly covered tubes) and centrifuged for min at.000rpm. Remove supernatant and left approximately ul - 0uL in the bottom of the tube. Add 0uL of 0% ethanol to each tube. (Grade PA - UPS / BIOTECHNOLOGICAL / MOLECULAR BIOLOGY / NO USE TECHNICAL GRADE). Centrifuge for min at.000rpm. Remove all supernatant. Drie all tubes in thermoplate between 0-0 C for minute. When there is no ethanol in the tubes, resuspend the pellet in 0-0uL of DNA Rehydration solution. Add ul of Template to each tube with PCR Premixture (see table in procedure manual). Add to each tube a drop of mineral oil. Run PCR program (see program in procedure manual). min min

7 DNA extraction protocol from swabs Label ml tubes with respective samples. Cut the tip swabs and insert in tubes. Add 0,mL of nucleolysis solution to each tube. Add ul Proteinase K to each tube. Perform Vortex cycles for seconds. Add ul RNAse and incubate 0min in thermoregulated bath between 0 and C. Dry thoroughly each tube and perform Spin Down. to retrieve drops of water evaporated. Remove the torula fragment of each tube. Add 0uL of Protein Precipitation solution. Perform Vortex cycles of seconds. Centrifuge at.000rpm for min. Take 00uL supernatant and move to new tube containing 00uL of Isopropanol (grade PA - UPS / BIOTECHNOLOGICAL / MOLECULAR / NO USE TECHNICAL GRADE BIOLOGY), discard the remainder. In new tube Observe DNA ring on top of the solution. Homogenize manually times gently (remember. tightly covered tubes) and centrifuged for min.000rpm. Remove supernatant and left approximately. ul - 0uL in the bottom of the tube. Add 0uL of 0% ethanol to each tube. (Grade PA - UPS / BIOTECHNOLOGICAL / MOLECULAR BIOLOGY / NO USE TECHNICAL GRADE). Centrifuge for min at.000rpm. Remove all supernatant. Drie all tubes in thermoplate between 0-0 C for minute. When there is no ethanol in the tubes, resuspend the pellet in 0-0uL of DNA Rehydration solution. Add ul of Template to each tube with PCR Premixture (see table in procedure manual). Add to each tube a drop of mineral oil. Run PCR program (see program in procedure manual).

8 DNA extraction protocol from lee Add 0. - g stool sample in a sterile ml tube. Add to ml X PBS, homogenizes. Transfer 00 ul of the mixture to a new micro tube. Centrigue at,000 rpm for minute. Then discard the supernatant. Add 00 ul of PBS X and homogenize. Centrigue at,000 rpm for minute. Then discard the supernatant. Add 0,mL of nucleolysis solution to each tube. Add ul Proteinase K to each tube. Perform cycles s in Vortex. Add ul RNAse and incubate 0min in thermoregulated bath between 0 and C. Add ul of Protein Precipitation solution (/ volume, Vol x 0.). Perform cycles of s at Vortex. Centrifuge at.000rpm x min. Take 00uL of the supernatant and move to new tube containing 00uL of Isopropanol (grade PA - UPS / BIOTECHNOLOGICAL / MOLECULAR / NO USE TECHNICAL GRADE BIOLOGY), discard the remainder. In new tube Observe DNA ring on top of the solution. homogenize manually times gently (remember tightly covered tubes) and centrifuged for min.000rpm. The supernatant is removed and a volume of is left - 0uL in the bottom of the tube. Add 0uL of 0% ethanol to each tube. (Grade PA - UPS / BIOTECHNOLOGICAL / MOLECULAR BIOLOGY / NO USE TECHNICAL GRADE) Centrifuge for min.000rpm. All supernatant is removed. All tubes are dried to 0-0 C in plate for minute. When there is no ethanol in the tubes, the pellet is resuspended in 0-0uL of DNA Rehydration solution. Add ul PCR Mix in PCR tubes for each pathogen. Add ul Template into each PCR tube. Add to each tube a drop of mineral oil. Run PCR with the given program.