Description

This kit is used for eccDNA purification and amplification. It has the
following features:

1. All-in-one kit, the users don' t need to optimize every components.

2. Applicable to various types of cells, including cell culture, fresh tissue, frozen cells, blood cells.

3. Minimized chromosomal DNA and linear DNA contamination.

4. High recovery rate of eccDNA.

5. 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.

6. High sensitivity, a few dozen of reverse transcriptase molecules can be detected.

7. eccDNA can be further enriched by dye-based real-time RCA.

8. No exogenous nucleic acid is introduced into the samples.

9. Enough for 25 purification and amplification experiments, one experiment need 2E7 cells as starting materials.

10. Research use only, not for diagnostic use.

Kit Component

230222pt1, Large Box, Shipping and Storage at Ambient Temperatur.

No.	Component and Specification
1	Solution A 230222a, 50 mL in 60mL natural color plastic bottle
2	Solution B 230222b, 5 mL in 5 mL natural color plastic bottle
3	Solution B 230222b, 5 mL in 5 mL natural color plastic bottle
4	Solution F 230222f, 100 mL in 100 mL brownglassbottle
5	Solution G 230222g, 100 mL in 100 mL brownglassbottle
6	eccDNA Precipitation Solution 221231, 110 mL in 120 mL natural color plastic bottle
7	User Manual, 230222sc, 1 set

230222pt2, 10-Wells Plastic Box, Shipping in cold and Stored at -20 ssd.

No.	Component and Specification
1	SolutionK 220137, 0.5 mL in 0.5 mL yellow-capped tube
2	Ultrapure Water 210806, 1 mL in 1.5 mL blue-capped tube
3	Human mtDNA Depletion A 230222h, 0.5 mL in 0.5 mL green-capped tube
4	Human mtDNA Depletion B 230222i, 0.5 mL in 0.5 mL purple-capped tube
5	RCA Primer 220225, lyophilized powder in 0.5 mL white-capped tube
6	RCA Reaction Buffer 221198b, 80 μL in 0.5 mL green-capped tube
7	RCA DNA Polymerase 221198a, 30 μL in 0.5 mL red-capped tube

Shipping and Storage

Shipping at low temperature, store Part 1 at 4 ℃ and Part 2 at -20 ℃, the storage period is 12 months

Materials Required but not Supplied in the Kit

Sample, PBS, ethanol

Procedures

Section 1: Purificationof eccDNA including mtDNA

1. 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.

2. 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.

3. 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.

4. Add 0.1 mL cell suspension into the small container of eachcaps.

5. Add 0.1 mL Solution E and mix it gently with the cells added in previous step using a pipette tip.

6. Repeat this process until all ten caps are filled with the cell-Solution C mixture.

7. Put the caps at 4℃ for 30 minutes and the mixtures will become jelly blocks.

8. Pry up the ten jelly blocks using a pipette tip and transfer them into two 1.5mL microcentrifuge tubes, 5 pieces/each.

9. Into each tubes, add 1mL Solution A, 0.1mL Solution B and 10 μL Solution K.

10. Gently shake the two tubes for 12 hours on a destaining shaker at room temperature.

11. Transfer the liquid in the two tubes into four microcentrifuge tubes, about 0.5 mL/each.

12. 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.

13. 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.

14. 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.

15. Add 1 mL 75% ethanol to the pellet, inverted 10 times and spin at 13,000×g for 3 minutes, discard the supernatant.

16. Spin at 13,000×g for 0.5 minutes at room temperature, discard the residual supernatant.

17. 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.

Section 2: human mtDNA Depletion (only for human eccDNA, For DNA from other species, this sectionof steps can be skipped)

18. 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.

19. 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.

20. 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.

21. 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.

22. Add 1 mL 75% ethanol to the pellet, inverted 10 times and spin at 13,000×g for 5 minutes, discard the supernatant.

23. Spin at 13,000×g for 0.5 minutes at room temperature, discard the residual supernatant.

24. Add 50 μL ultrapure water to dissolve the pellet. The resultant solution is mtDNA-depleted eccDNA solution.

Section 3: RCA of eccDNA

25. 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.

26. Set up RAC reactions according to the following table:

Component	Sample Tube	Negative Control Tube
eeccDNA from step 21	5.5μL	-
Ultrapure Water	-	5.5μL
RCA Primer Mix	1μL	1μL
RCA Reaction Buffer	2.5μL	2.5μL
Total	9μL	9μL

27. 95℃ 5 minutes and put on ice immediately.

28. Add 1μL RCA DNA Polymerase and mix gently.

29. Incubate at 30℃for 16 hours on a real-time PCR machine, collecting signal in the SYBR channel in every 60 seconds.

30. 95℃ 5 minutes to inactive the enzyme and the amplification products can be used for further analysis or stored for later use.

Documents

User Manual

eccDNA Purification and Amplification Kit (Embedding Method)