Simplified library construction method and reagent
A library construction and fragmentation technology, applied in the field of simplified library construction methods and reagents, can solve the problems of inability to integrate the fragmentation and end repair steps, increase the loss of DNA samples, and take a long time to meet the requirements of sequencing on-machine, The effect of reducing DNA loss
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Embodiment 1
[0039] Embodiment 1, a kind of simplified library construction method
[0040] A simplified library construction method of the present invention, the specific process is as follows:
[0041] (1) Fragmentation and end repair
[0042] Prepare reaction solution A (10X) for fragmentation and end-repair one-step reaction, the formula is as follows:
[0043]
[0044]
[0045] Configure the following system:
[0046]
[0047] Wherein, enzyme mixture B includes fragmentation enzyme (NEB Company) and end repair enzyme (Life Company), and the two are mixed at a volume ratio of 1:1.
[0048] After mixing, carry out the following reaction, and store at 4°C after the reaction:
[0049]
[0050] (2) Ligation of sequencing adapters
[0051] For the adapter ligation of the product after the previous step reaction, add the following reagents:
[0052]
[0053] Among them, the P1 adapter is a P1-Adapter composed of the following two positive and negative sequences:
[0054]...
Embodiment 2
[0061] A simplified library construction method, after the step (1) in Example 1, the "magnetic bead purification" step is added, and other steps remain unchanged. Among them, the supplementary magnetic bead purification steps are as follows: Purify the fragmented and end-repaired product with 36 μL of XP magnetic beads for 5 minutes, place it on the magnetic stand for 3 minutes, then aspirate the liquid, wash twice with 500 μL of 75% ethanol, and blot the residue. Dry the liquid, add 20 μL Low TE solution, mix well, let it stand for 5 minutes, absorb the liquid, and it will be the purified fragmentation and end repair product, which is used to connect the sequencing adapter.
Embodiment 3
[0063] A simplified library construction method. In step (1) of Example 1, the formula of reaction solution A (10X) is optimized. The formulas of each group are as follows, and other steps remain unchanged:
[0064]
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