5 min Circulating DNA Extraction Kit

Transcription

1 5 min Circulating DNA Extraction Kit Biofactories 5 min Circulating DNA Extraction Kit provides the fastest method for the isolation and purification of circulating or cell free DNA from serum, plasma, or urine. The kit is specifically designed for effective purification of small fragmented DNA from the listed samples. Analyses of circulating or cell free DNA is essential step in several diagnosis such as SNP analyses for drug response and mutational studies in BRCA 1, 2, k-ras, EGFR, etc. Detection of somatic mutations would be very powerful tool in early cancer diagnosis. Moreover, the circulating DNA analyses can be a sensitive, safe, and alternative test for fetus genetic alternation of Trisomy or Down syndrome. The recovery of good quality Circulating DNA from limited volume of samples can be quite challenging because of several reasons. First, the amount of circulating DNA in sample is highly limited. Second the specimen contains high amount of serum protein that may inhibit for selective isolation of DNA. Therefore, a reliable method is required for extraction of circulating or cell free DNA from serum, plasma, or urine. Circulating DNA or cell free DNA can be extracted in less than 5 minutes by 5 min Circulating DNA Extraction Kit. For efficient extraction, the lysis solution is designed based on a unique chemistry for most effective binding and recovery. Also the DNA can be eluted in a flexible volume of 25 to 100 ul. Instead of more than 3 hours of procedure by other products, you will see the same to similar recovery of DNA in less than 5 minute. The purified DNA is of the highest integrity, and can be used in a number of downstream applications such as PCR and qpcr. 5 min Circulating DNA Extraction Kit

2 Catalog #; D-1009 The fastest and simplest but most reliable Nucleic Acid extraction system Purification of cell free DNA or circulating DNA from plasma, serum, or urine Total procedure in less than 5 minutes not in 2-3 hours like other products. Can handle up to 5 ml of serum, plasma, or urine Single column format Monitoring of cfdna amount and/or detection of cfdna originated from cancer cell would be sensitive assay tool for early cancer diagnosis One kit for 50 DNA purification The extracted DNA is not applicable to Nanodrop reading because of low amount. The extracted DNA can be used in downstream steps of qpcr and PCR without any inhibitory effect 5 min Circulating DNA Extraction Kit

3 Storage Conditions and Product Stability All components should be kept tightly sealed and stored at room temperature. These reagents should remain stable for at least 1 year in their unopened containers. Precautions and Disclaimers This kit is designed for research purposes only. It is not intended for human or diagnostic use. Customer-Supplied Reagents and Equipment Benchtop microcentrifuge 1.5 ml microcentrifuge tubes Vortex % ethanol % isopropanol Kit components for 1 box/50 prep Component Column/collection tube Circulating DNA Solution DNA Washing Solution DNA Elution buffer Product insert Products 50 ea 46 ml 18 ml 7.5 ml 1 ea 5 min Circulating DNA Extraction Kit

4 5 min Circulating DNA Extraction Kit Before Starting 1. Add 42 ml of % ethanol to DNA Washing Solution. Mix well. Mark on the labels that ethanol is added. Store both wash buffers with ethanol at room temperature. 2. Add 17ml of % isopropanol to Circulating DNA Solution. Mix well. Mark on the labels that ethanol is added. Store both wash buffers with ethanol at room temperature. Plasma preparation Notice Blood in EDTA tube should be used. Plasma preparation should be done within an hour of blood drew. If the preparation is delayed, cell free DNA or circulating DNA would be contaminated with genomic DNA released from white blood cell. Procedure 1. Centrifuge the Blood EDTA tube at 5,000 rpm for 2 min using a clinical centrifuge. 2. Harvest all plasma into 1.5 ml microtube and spin at 13,500 rpm for 5 min. 3. Carefully remove supernatant without interference of the pellet. 4. Freeze at -20 o C until use. 5 min Circulating DNA Extraction Kit 4

5 Procedures for 5 min Circulating DNA extraction from less than 0.5 ml of plasma or urine Important notice; All centrifugation in 10,000 rpm 1. Add less than 0.5 ml of plasma or urine sample to 1.5 ml of microtube. 2. Add 2 times volume of Circulating DNA Solution and vortex for 30 sec. 3. Transfer 700 ul of the lysate to column. 4. Centrifuge for 15 sec. 5. Discard the flowthrough. Reassemble the spin column with its collection tube. 6. Repeat steps 3 to 5 as required. 7. Apply 700 ul of DNA Washing Solution and centrifuge for 15 sec. Discard the flowthrough. 8. Apply 400ul of DNA Washing Solution and centrifuge for 30 sec. Discard the flowthrough. 9. Replace the collection tube with a clean microcentrifuge tube (not supplied). 10. Add ul of DNA Elution Buffer to column. 11. Close the cap and vortex for 15 sec. 12. Centrifuge for 1 min. 5 min Circulating DNA Extraction Kit 5

6 Procedures for 5 min Circulating DNA extraction from more than 0.5 ml of plasma or urine Important notice; All centrifugation in 10,000 rpm 1. Add less than 5 ml of plasma or urine sample to 15 ml conical tube. 2. Add 2 times volume of Circulating DNA Extraction Solution and vortex for 30 sec. 3. Transfer 700 ul of the lysate to column. 4. Centrifuge for 15 sec. 5. Discard the flowthrough. Reassemble the spin column with its collection tube. 6. Repeat steps 3 to 5 as required. 7. Apply 700 ul of DNA Washing Solution and centrifuge for 15 sec. Discard the flowthrough. 8. Apply 400ul of DNA Washing Solution and centrifuge for 30 sec. Discard the flowthrough. 9. Replace the collection tube with a clean microcentrifuge tube (not supplied). 10. Add 100ul of DNA Elution Buffer to column. 11. Close the cap and vortex for 15 sec. 12. Centrifuge for 1 min. 5 min Circulating DNA Extraction Kit 6

7 Example of test Circulating DNA was extracted from the indicated volume of 5 different plasma samples using 5 min Circulating DNA Extraction Kit or Qiamp Circulating DNA Kit. Each DNA was eluted final 100 ul. Total amount of circulating DNA was quantitated by qpcr using human actin primers. Each number indicates the average of two independent assays by CFX96 (Biorad). Sample ID and used volume Qiamp Kit 5 min Kit 1; 0.5 ml ; 0.5 ml ; 0.5 ml ; 0.5 ml ; 0.5 ml ; 0.25 ml ; 0.25 ml ; 0.25 ml ; 0.25 ml ; 0.25 ml Extracted circulating DNA from 8 independent plasma samples was used for PCR reaction for EGFR detection. DNA was extracted from 250 ul of plasma samples and eluted to final 100 ul. Five ul of each sample was used for the PCR reaction. The size of PCR product is 240 bp. 5 min Circulating DNA Extraction Kit 7

8 Circulating DNA was extracted from the indicated volume of plasma samples using 5 min Circulating DNA Extraction Kit. Each DNA was eluted final 100 ul. Total amount of circulating DNA was quantitated by qpcr using human actin primers. Each number indicates the average of two independent assays by CFX96 (Biorad). Plasma volume (ml) hactin Ct value Circulating DNA was extracted from 0.5ml of five different urine samples using 5 min Circulating DNA Extraction Kit. Each DNA was eluted in final 100 ul. Total amount of circulating DNA was quantitated by qpcr using human actin primers and SYBR 2 X mix from Bioline. The analyses were done by Smartcycler from Cepheid. 5 min Circulating DNA Extraction Kit 8

9 Ct value Tm Technical Support Biofactories Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of our products. If you have any questions or experience any difficulties regarding products, please do not hesitate to contact us. For technical assistance and more information, please contact our Technical Support Team through at 5 min Circulating DNA Extraction Kit 9

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