The CD scWGS MDA Amplication Kit, based on multiple chain displacement amplifications, provides a quick and easy workflow for whole genome amplification from single cells and other micro samples (MDA). This kit can produce 30-40G amplification products from 1-1000 cells or 1ng-10ng purified DNA as a framework for whole-genome sequencing using 4L cell specimens and 2.5L purified DNA specimens. The kit includes all of the enzymes and buffers required for cell lysis and whole genome amplification. Amplification products vary in size from 2 kb to 100 kb, with a 95 percent coverage rate and an average length of more than 20 kb. Phi29 DNA polymerase is used in multiple displacement amplification (MDA), guaranteeing DNA amplification with an increased coverage rate, uniformity, sensitivity, and fidelity. It has a strong strand displacement activity, a strong chain affinity, and a potent 3'5 exonuclease (proofreading) activity. All kit elements have undergone rigorous quality checks to have a library preparation that is consistent and repeatable.
Storage:
All components should be stored at -20℃.
Components:
Specifications:
| Sample amount | 1-1000 of cells or 1ng-10ng of purified DNA |
| Features | High sensitivity: It can simplify whole genome from single cell or very minor amount of genome sample. High coverage rate: The coverage rate of single cell genome can be more than 95% after amplification. High uniformity: Non-preference based DNA amplification can ensure uniform amplification of whole genome. High fidelity: Phi29 DNA polymerase has higher fidelity than current major polymerases. |
| Application | It is recommended to use this kit for: Analysis of inter-cellular genome heterogeneity Mutation detection Copy number variation analysis |
| Species Category | animals, plants, Bacteria |
| Sample type | cells without cell wall or purified DNA |
| Sequencing Platform | Illumina |
Amplify genomic DNA from single cell
This scheme is suitable for 1-1000 initial cells. Please use the freshly prepared cell samples to ensure the integrity of initiated genome and do not use apoptotic cells.
1. Prepare Buffer D2.
2. Add 4 μl of cell samples (resuspended in PBS) to a PCR tube. Please make up to 4 μl with PBS if the sample volume is less than 4 μl.
3. Add 3 μl of Buffer D2. Flick the tube wall to mix the cells followed with briefly centrifugation.
Note: Please make sure that the cells are not attached to the tube wall. DO NOT mix the cells with pipettors, to avoid that the cells are attached to the tips.
4. Incubate at 65°C for 10 min.
5. Add 3 μl of Buffer N, and mix by flick the tube wall followed with briefly centrifugation. Place the sample on the ice before the next reaction is prepared.
6. Prepare the mixture of reaction.
7. Immediately add 40 μl of reaction mixture to the prepared 10 μl of DNA sample (prepared in the Step 5), mix thoroughly and briefly centrifugate.
8. Incubate at 30°C for 6 hours.
Note: Incubation for 16 hour can obtain the maximum yield. Please prolong the incubation time if necessary.
9. Incubate at 65°C for 5 min to inactivate the scDNA Polymerase.
10. The amplification product is high concentration genomic DNA, please dilute the DNA to an appropriate concentration with water or TE to perform the downstream experiment. The amplification products can be widely used in downstream experiment including whole genome sequencing, whole exome sequencing, microsatellite analysis, qPCR analysis, gene chip analysis, CGH Array, etc.
Amplify purified genomic DNA
1. Prepare Buffer D1 and N1.
2. Add 2.5 μl of cell samples to a PCR tube. If the sample volume is lower than 2.5 μl, please make up to 2.5 μl with PBS.
3. Add 2.5 μl of Buffer D1. Flick the tube wall to mix the cells followed with briefly centrifugation.
Note: DO NOT mix the cells with pipettors, to avoid that the integrity is affected and the cells are attached to the tips.
4. Incubate at 15°C-25°C for 3 min.
5. Add 5 μl of Buffer N1. Flick the tube wall to mix the cells followed with briefly centrifugation. Place the sample on the ice before the next step.
6. Prepare the mixture of reaction.
7. This Immediately add 40 ul of reaction mixture to 10 ul of DNA sample (prepared in Step 5), flick the tube wall to mix the cells followed with briefly centrifugation.
8. Incubate at 30°C for 6 hours.
Note: Incubate for 16 hours can obtain the maximum yield. Please prolong the incubation time if necessary.
9. Incubate at 65°C for 5 min to inactivate the scDNA Polymerase.
10. The amplification product is high concentration genomic DNA, please dilute the DNA to an appropriate concentration with water or TE to perform the downstream experiment. The amplification products can be widely used in downstream experiment including whole genome sequencing, whole exome sequencing, microsatellite analysis, qPCR analysis, gene chip analysis, CGH Array, etc.
For protocols on more sample types, please download the CD scWGS MDA Amplication Kit Handbook.
