Wash solution and washing method for hybrid-enrichment-capture DNA sequencing library
View Patent ↗This application provides a detailed description of a washing solution consisting of saline sodium citrate (SSC) buffer and sodium dodecyl sulfate (SDS) for hybridization-enrichment-capture DNA sequencing libraries as well as a corresponding washing method. The invented solution has a simple recipe, which is low-cost and easily produced and stored. The washing strategy associated with the invented solution is simple and easy to carry out, requiring no special instruments, and the results produced thereby are outstanding with low background noise, which consequently leads to an improvement on the efficiency of targeted enrichment. Elution of the library after washing is easy to carry out and requires no additional reagents or purification, effectively reducing loss of material due to further purification and time needed for completion of procedure.
1. A washing solution for hybridization-enrichment-capture DNA sequencing libraries, wherein the washing solution includes the following three portions:
a) washing solution I: 1×SSC, 0.1% SDS;
b) washing solution II: 0.1×SSC, 0.1% SDS;
c) washing solution III: 0.2×SSC, 10% SDS,
wherein the washing solutions I, II and III each consist of saline sodium citrate (SSC) buffer and sodium dodecyl sulfate (SDS), and the washing solutions I, II and III are separately packaged.
2. A method for washing hybridization-enrichment-capture DNA sequencing libraries by using a washing solution including following three portions:
a) washing solution I: 1×SSC, 0.1% SDS;
b) washing solution II: 0.1×SSC, 0.1% SDS;
c) washing solution III: 0.2×SSC, 10% SDS,
wherein the SSC consists of 3M NaCl and 300 mM sodium citrate with a pH value of 7.0,
the method comprising the following steps:
(1) for each enrichment reaction, pre-incubating 1 ml washing solution I and 2 ml washing solution II at 65° C. for 30 minutes, and placing the other two portions under room temperature;
(2) briefly centrifuging a hybridization reaction with biotin-labeled DNA probes captured by Invitrogen Dynabeads® M270 Streptavidin beads in a centrifuge tube at 600×g for 3 seconds, to ensure that there are no beads left on the tube wall or tube lid;
(3) placing the centrifuge tube on a magnetic separation rack and letting it stand for 1 minute until the liquid inside is clear, then carefully removing and discarding the supernatant;
(4) removing the centrifuge tube from magnetic stand and adding 1 ml of washing solution I pre-warmed at 65° C. in step (1), mixing thoroughly by pipetting up and down for 10 times, and then incubating the mixture at 65° C. for 5 minutes;
(5) placing the centrifuge tube on the magnetic separation rack and letting it stand for 1 minute until the liquid inside is clear, then carefully removing and discarding the supernatant;
(6) removing the centrifuge tube from magnetic stand and adding 1 ml of washing solution II pre-warmed at 65° C. in step (1), mixing thoroughly by pipetting up and down for 10 times, and then incubating the mixture at 65° C. for 5 minutes;
(7) placing the centrifuge tube on the magnetic separation rack and letting it stand for 1 minute until the liquid inside is clear, then carefully removing and discarding the supernatant;
(8) repeating step (6) to (7) for one more time;
(9) removing the centrifuge tube from magnetic stand and adding 1 ml of room temperature washing solution II, mixing thoroughly by pipetting up and down for 10 times, and then placing the centrifuge tube into rotary mixer and spinning it for 5 minutes;
(10) removing the centrifuge tube from magnetic stand and briefly centrifuging it at 600×g for 3 seconds to ensure that no beads are left on the tube wall or tube lid;
(11) placing the centrifuge tube on the magnetic separation rack and letting it stand until the liquid inside is clear, then carefully removing and discarding the supernatant as much as possible with a P10 pipette;
(12) keeping the centrifuge tube on the magnetic rack and adding 1 ml of washing solution III against the tube wall opposite to the beads, counting 30 seconds then carefully removing and discarding the supernatant as much as possible with the pipette;
(13) letting the beads dry for 2 minutes, adding 22.5 μl nuclease-free water to resuspend magnetic beads by mixing them with the pipette up and down for 10 times, followed by heating the centrifuge tube at 98° C. for 10 minutes;
(14) after heating, vortex mixing the centrifuge tube and then briefly centrifuging it at 600×g for 3 seconds to ensure that no beads are left on the tube wall or tube lid;
(15) placing the centrifuge tube on the magnetic rack until the liquid inside is clear, immediately removing 20 μl supernatant containing hybridization-capture enriched DNA library sample for subsequent post-enrichment amplification.