DNA extraction protocol for snake genomic sequencing by using AxyPrepTM Multisource Genomic DNA Miniprep Kit
Boyang Liu, Liangyu Cui, Yue Ma, Diancheng Yang, Yanan Gong, Yanchun Xu, Shuhui Yang, Song Huang, Zhangwen Deng
Abstract
Genomic DNA was extracted from the muscle of a snake using the AxyPrepTM Multisource Genomic DNA Miniprep Kit (Axygen, China), with some modifications from the kit instructions as outlined in this protocol. The extracted DNA was then suitable for WGS sequencing.
Steps
The following protocol is a modified DNA extraction protocol using the AxyPrepTM Multisource Genomic DNA Miniprep Kit.
Approximately 10mg of snake tissue was minced into small pieces using sterilized scissors on ice.
On ice
Add 350 µl Buffer PBS and 15 µl SDS Lysis Buffer and shake for 30s.
0h 0m 30s
Add 150 µl Buffer C-L and 20 µl Proteinase K. Immediately vortex for 1 min to mix well. After brief centrifugation, incubate at 56°C for 10 min.
0h 1m 0s
56°C 0h 10m 0s
Add 350 µl Buffer P-D, vortex for 30s to mix well, and centrifuge at 12,000×g for 10 min.
0h 0m 30s
12000rpm
Place the DNA preparation tube in a 2 ml centrifuge tube, transfer the mixture in step 4 to the preparation tube, and centrifuge at 12,000×g for 1 min.
12000rpm
Discard the filtrate, put the preparation tube back into the original 2 ml centrifuge tube, add 500µl Buffer W1, and centrifuge at 12,000×g for 1 min.
12000rpm
Discard the filtrate, put the preparation tube back into the original 2 ml centrifuge tube, add 700µl Buffer W2, centrifuge at 12,000×g for 1 min.
12000rpm
In the same way, cleanse again with 700µl Buffer W2.
Discard the filtrate, put the preparation tube back into the original 2ml centrifuge tube, and centrifuge at 12,000×g for 1 min.
12000rpm
Place the DNA preparation tube in another clean 1.5 ml centrifuge tube, add 100-200µl Eluent or deionized water to the center of the preparation tube membrane, stand at room temperature for 1 min, and centrifuge at 12,000×g for 1 min to elute the DNA.
0h 1m 0s Room temperature
12000rpm
