{"product_id":"eccdna-purification-and-amplification-kit","title":"eccDNA Purification and Amplification Kit (Embedding Method)","description":"\u003ch2\u003eDescription\u003c\/h2\u003e\n\u003cp\u003eThis kit is used for eccDNA purification and amplification. It has the \u003cbr\u003efollowing features:\u003c\/p\u003e\n\u003cp\u003e1. All-in-one kit, the users don' t need to optimize every components.\u003c\/p\u003e\n\u003cp\u003e2. Applicable to various types of cells, including cell culture, fresh tissue, frozen cells, blood cells.\u003c\/p\u003e\n\u003cp\u003e3. Minimized chromosomal DNA and linear DNA contamination.\u003c\/p\u003e\n\u003cp\u003e4. High recovery rate of eccDNA.\u003c\/p\u003e\n\u003cp\u003e5. Specific depletion of human mitochondrial DNA, which is cccDNA. For samples from species other than human, the mitochondrial DNA can only be deleted via bioinformatic means.\u003c\/p\u003e\n\u003cp\u003e6. High sensitivity, a few dozen of reverse transcriptase molecules can be detected.\u003c\/p\u003e\n\u003cp\u003e7. eccDNA can be further enriched by dye-based real-time RCA.\u003c\/p\u003e\n\u003cp\u003e8. No exogenous nucleic acid is introduced into the samples.\u003c\/p\u003e\n\u003cp\u003e9. Enough for 25 purification and amplification experiments, one experiment need 2E7 cells as starting materials.\u003c\/p\u003e\n\u003cp\u003e10. Research use only, not for diagnostic use.\u003c\/p\u003e\n\u003ch2\u003eKit Component\u003c\/h2\u003e\n\u003cp\u003e\u003cstrong\u003e230222pt1, Large Box, Shipping and Storage at Ambient Temperatur.\u003c\/strong\u003e\u003c\/p\u003e\n\u003ctable style=\"width: 90%; border-collapse: collapse;\" border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 16.6998%;\"\u003eNo.\u003c\/td\u003e\n\u003ctd style=\"width: 83.3002%; text-align: center; vertical-align: middle;\"\u003eComponent and Specification\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 16.6998%;\"\u003e1\u003c\/td\u003e\n\u003ctd style=\"width: 83.3002%;\"\u003eSolution A\u003cbr\u003e230222a, 50 mL in 60mL natural color plastic bottle\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 16.6998%;\"\u003e2\u003c\/td\u003e\n\u003ctd style=\"width: 83.3002%;\"\u003eSolution B\u003cbr\u003e230222b, 5 mL in 5 mL natural color plastic bottle\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 16.6998%;\"\u003e3\u003c\/td\u003e\n\u003ctd style=\"width: 83.3002%;\"\u003eSolution B\u003cbr\u003e230222b, 5 mL in 5 mL natural color plastic bottle\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 16.6998%;\"\u003e4\u003c\/td\u003e\n\u003ctd style=\"width: 83.3002%;\"\u003eSolution F\u003cbr\u003e230222f, 100 mL in 100 mL brownglassbottle\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 16.6998%;\"\u003e5\u003c\/td\u003e\n\u003ctd style=\"width: 83.3002%;\"\u003eSolution G\u003cbr\u003e230222g, 100 mL in 100 mL brownglassbottle\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 16.6998%;\"\u003e6\u003c\/td\u003e\n\u003ctd style=\"width: 83.3002%;\"\u003eeccDNA Precipitation Solution\u003cbr\u003e221231, 110 mL in 120 mL natural color plastic bottle\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 16.6998%;\"\u003e7\u003c\/td\u003e\n\u003ctd style=\"width: 83.3002%;\"\u003eUser Manual, 230222sc, 1 set\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003e230222pt2, 10-Wells Plastic Box, Shipping in cold and Stored at -20 ssd.\u003c\/strong\u003e\u003c\/p\u003e\n\u003ctable border=\"1\" style=\"width: 90%; border-collapse: collapse;\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 10%;\"\u003eNo.\u003c\/td\u003e\n\u003ctd style=\"width: 50%; text-align: center; vertical-align: middle;\"\u003eComponent and Specification\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 10%;\"\u003e1\u003c\/td\u003e\n\u003ctd style=\"width: 50%;\"\u003eSolutionK\u003cbr\u003e220137, 0.5 mL in 0.5 mL yellow-capped tube\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 10%;\"\u003e2\u003c\/td\u003e\n\u003ctd style=\"width: 50%;\"\u003eUltrapure Water\u003cbr\u003e210806, 1 mL in 1.5 mL blue-capped tube\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 10%;\"\u003e3\u003c\/td\u003e\n\u003ctd style=\"width: 50%;\"\u003eHuman mtDNA Depletion A\u003cbr\u003e230222h, 0.5 mL in 0.5 mL green-capped tube\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 10%;\"\u003e4\u003c\/td\u003e\n\u003ctd style=\"width: 50%;\"\u003eHuman mtDNA Depletion B\u003cbr\u003e230222i, 0.5 mL in 0.5 mL purple-capped tube\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 10%;\"\u003e5\u003c\/td\u003e\n\u003ctd style=\"width: 50%;\"\u003eRCA Primer\u003cbr\u003e220225, lyophilized powder in 0.5 mL white-capped tube\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 10%;\"\u003e6\u003c\/td\u003e\n\u003ctd style=\"width: 50%;\"\u003eRCA Reaction Buffer\u003cbr\u003e221198b, 80 μL in 0.5 mL green-capped tube\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align: center; vertical-align: middle; width: 10%;\"\u003e7\u003c\/td\u003e\n\u003ctd style=\"width: 50%;\"\u003eRCA DNA Polymerase\u003cbr\u003e221198a, 30 μL in 0.5 mL red-capped tube\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003ch2\u003eShipping and Storage \u003c\/h2\u003e\n\u003cp\u003eShipping at low temperature, store Part 1 at 4 ℃ and Part 2 at -20 ℃, the storage period is 12 months\u003c\/p\u003e\n\u003ch2\u003eMaterials Required but not Supplied in the Kit\u003c\/h2\u003e\n\u003cp\u003eSample, PBS, ethanol\u003c\/p\u003e\n\u003ch2\u003eProcedures\u003c\/h2\u003e\n\u003cp\u003e\u003cstrong\u003eSection 1: Purificationof eccDNA including mtDNA\u003c\/strong\u003e\u003c\/p\u003e\n\u003cp\u003e1. Collect cells of interest from cell culture, fresh tissue, frozen tissue and blood using appropriate methods, wash the cells thrice with ice cold PBS, resuspend the cells at a final concentration of 2E7 cells\/mL with PBS in a 1.5 mL microcentrifuge tube. Put the tube in a heat block set at 42℃ until use.\u003c\/p\u003e\n\u003cp\u003e2. Put the bottle that contains Solution E in 80℃ water bath until Solution Eis well melted. Transfer 1 mL melted Solution Eto a 1.5 mL microtube and put the tube in a heat block set at 42℃ until use.\u003c\/p\u003e\n\u003cp\u003e3. Cut off the caps of ten 1.5 mL microcentrifuge tubes and put the up flat sides down on the lab bench, with the inward part of the cap facing upward.\u003c\/p\u003e\n\u003cp\u003e4. Add 0.1 mL cell suspension into the small container of eachcaps.\u003c\/p\u003e\n\u003cp\u003e5. Add 0.1 mL Solution E and mix it gently with the cells added in previous step using a pipette tip. \u003c\/p\u003e\n\u003cp\u003e6. Repeat this process until all ten caps are filled with the cell-Solution C mixture. \u003c\/p\u003e\n\u003cp\u003e7. Put the caps at 4℃ for 30 minutes and the mixtures will become jelly blocks.\u003c\/p\u003e\n\u003cp\u003e8. Pry up the ten jelly blocks using a pipette tip and transfer them into two 1.5mL microcentrifuge tubes, 5 pieces\/each.\u003c\/p\u003e\n\u003cp\u003e9. Into each tubes, add 1mL Solution A, 0.1mL Solution B and 10 μL Solution K.\u003c\/p\u003e\n\u003cp\u003e10. Gently shake the two tubes for 12 hours on a destaining shaker at room temperature. \u003c\/p\u003e\n\u003cp\u003e11. Transfer the liquid in the two tubes into four microcentrifuge tubes, about 0.5 mL\/each.\u003c\/p\u003e\n\u003cp\u003e12. Add 0.2 mL Solution F to each tubes and vortex for 2 minutes, spin at 13,000×g for 5 minutes at room temperature, transfer the upper layer to a new microcentrifuge tube.\u003c\/p\u003e\n\u003cp\u003e13. Add 0.2 mL Solution G to each tubes and vortex for 2 minutes, spin at 13,000×g for 5 minutes at room temperature, transfer the upper layer to a new microcentrifuge tube.\u003c\/p\u003e\n\u003cp\u003e14. Add 1 mL eccDNAPrecipitation Solution (shake to mix well before use) to each tube, invert 10 time to mixwell, spin at 13,000×g for 15 minutes at room temperature, discard the supernatant.\u003c\/p\u003e\n\u003cp\u003e15. Add 1 mL 75% ethanol to the pellet, inverted 10 times and spin at 13,000×g for 3 minutes, discard the supernatant.\u003c\/p\u003e\n\u003cp\u003e16. Spin at 13,000×g for 0.5 minutes at room temperature, discard the residual supernatant.\u003c\/p\u003e\n\u003cp\u003e17. Add 50 μL ultrapure water to dissolve the pellet of each tubes and the solution obtained is eccDNA solution which includes mitochondrial DNA. Pool the DNA solution from the 4 tubes in to one tube and save 20 μL for later use as the negative control for mitochondrial DNA depletion. The rest will be used in then ext step.\u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003eSection 2: human mtDNA Depletion (only for human eccDNA, For DNA from other species, this sectionof steps can be skipped)\u003c\/strong\u003e\u003c\/p\u003e\n\u003cp\u003e18. To 180 μL pooled DNA, add 20 μL Human mtDNA Depletion Aand 1 μL Human mtDNA Depletion B, mix well and incubate at 37℃for 4 hours.\u003c\/p\u003e\n\u003cp\u003e19. Add 0.2 mL Solution F and vortex for 2 minutes, spin at 13,000×g for 5 minutes at room temperature, transfer the upper layer to a new microcentrifuge tube.\u003c\/p\u003e\n\u003cp\u003e20. Add 0.2 mL Solution G and vortex for 2 minutes, spin at 13,000×g for 5 minutes at room temperature, transfer the upper layer to a new microcentrifuge tube.\u003c\/p\u003e\n\u003cp\u003e21. Add 200μL eccDNA Precipitation Solution (shake to mix well before use) to the upper layer, invert 10 time to mix, spin at 13,000×g for 15 minutes at room temperature, discard the supernatant.\u003c\/p\u003e\n\u003cp\u003e22. Add 1 mL 75% ethanol to the pellet, inverted 10 times and spin at 13,000×g for 5 minutes, discard the supernatant.\u003c\/p\u003e\n\u003cp\u003e23. Spin at 13,000×g for 0.5 minutes at room temperature, discard the residual supernatant.\u003c\/p\u003e\n\u003cp\u003e24. Add 50 μL ultrapure water to dissolve the pellet. The resultant solution is mtDNA-depleted eccDNA solution.\u003c\/p\u003e\n\u003cp\u003e\u003cstrong\u003eSection 3: RCA of eccDNA\u003c\/strong\u003e\u003c\/p\u003e\n\u003cp\u003e25. For the first time using the kit, add 25μL ultrapure water into the white-capped RCA Primer tube, vortex for 30 seconds and the resultant solution is RCA Primer Mix. Put the mix on ice for later use and at -80℃for long-term storage. \u003c\/p\u003e\n\u003cp\u003e26. Set up RAC reactions according to the following table:\u003c\/p\u003e\n\u003ctable border=\"1\" style=\"width: 90%; border-collapse: collapse;\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003eComponent\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003eSample Tube\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003eNegative Control Tube\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003eeeccDNA from step 21\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e5.5μL\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e-\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003eUltrapure Water\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e-\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e5.5μL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003eRCA Primer Mix\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e1μL\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e1μL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003eRCA Reaction Buffer\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e2.5μL\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e2.5μL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003eTotal\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e9μL\u003c\/td\u003e\n\u003ctd style=\"width: 33.3333%; text-align: center; vertical-align: middle;\"\u003e9μL\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\n\u003c\/table\u003e\n\u003cp\u003e \u003c\/p\u003e\n\u003cp\u003e27. 95℃ 5 minutes and put on ice immediately.\u003c\/p\u003e\n\u003cp\u003e28. Add 1μL RCA DNA Polymerase and mix gently.\u003c\/p\u003e\n\u003cp\u003e29. Incubate at 30℃for 16 hours on a real-time PCR machine, collecting signal in the SYBR channel in every 60 seconds. \u003c\/p\u003e\n\u003cp\u003e30. 95℃ 5 minutes to inactive the enzyme and the amplification products can be used for further analysis or stored for later use.\u003c\/p\u003e\n\u003ch2\u003eDocuments\u003cbr\u003e\n\u003c\/h2\u003e\n\u003cp\u003e\u003ca href=\"https:\/\/cdn.shopify.com\/s\/files\/1\/0521\/5312\/2997\/files\/eccDNA_Purification_and_Amplification_Kit_Embedding_Method.pdf?v=1750314480\" type=\"application\/pdf\" target=\"_blank\"\u003e\u003cstrong\u003eUser Manual\u003c\/strong\u003e\u003c\/a\u003e\u003c\/p\u003e","brand":"SkyGen","offers":[{"title":"25T","offer_id":51229010395317,"sku":"230222","price":2058.0,"currency_code":"USD","in_stock":true}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/0521\/5312\/2997\/files\/eccDNA_Purification_and_Amplification_Kit_Embedding_Method.png?v=1751005439","url":"https:\/\/biofargo.com\/products\/eccdna-purification-and-amplification-kit","provider":"Biofargo","version":"1.0","type":"link"}