Fast gel dna extraction mini kit

DNA libraries of these hornet venoms were built. We used the HiS-cript®II 1st Stand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd) to reverse transcribe RNA into first-strand cDNA. Double-strand cDNA was then synthesized by utilizing first-strand cDNA, a 5 ′ PCR primer: 5 ′ -AAGCAGTGGTATCAACGCAGAGT-3 ′ and the 3 ′ SMARTTM CDS III/3 ′ PCR Primer: 5 ′ -ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3' (N = A, G, C, or T; N-1 = A, G, or C) in the second-strand to allow long distance PCR. Finally, this cDNA was used as a template with a 5 ′ primer (5 ′ -ATGAGTGCCGAAGCTTTAGCT-3 ′ ) and the 3 ′ SMART™ CDS III/3 ′ PCR Primer for PCR. We used the following settings: 1 min at 94 • C, 30 cycles for 10 s at 92 • C, 30 s at 53 • C, and 40 s at 72 • C, followed by a final 10 min at 72 • C. We used a general rTaq enzyme (TaKaRa Biotechnology Co.Ltd., Dalian, China).
The resulting PCR amplification products were then purified with a Fast Gel DNA Extraction Mini Kit (Vazyme Biotech Co., Ltd). The purified cDNA was then linked to a pMD™19-T vector (TaKaRa Biotechnology Co. Ltd.), and the ligation products were inserted into incubated DH5a competent cells for cloning and sent to TsingKe Biotechnology Co. Ltd. (China) for cDNA sequencing.

After microinjection, wild-type and injected embryos were incubated and reared respectively in ltered seawater with continuous aeration at 22 ℃ until hatching. At 24 hpf, the genomic DNA was isolated from embryos by extraction solution and neutralization solution (Dongshengbio #9052). PCR ampli cation of targeted mstn gene was carried out with a system of 12.5 µL 2× Premix Taq (TaKaRa RR901), 8.5 µL RNase-free water, 1 µL primer F/R and 2 µL extracted DNA. The program was nished with the parameters showed in the Supplementary Table S 1. The PCR products were examined by 2% agarose gel electrophoresis using the Mini PROTEAN Tetra electrophoresis unit (Bio-Rad) and puri ed by Fast ® Gel DNA Extraction Mini kit (Vazyme, #DC301-01). To check if there was a mutation in the target region, the PCR ampli cation were cloned into the pMD TM 19-T vector (TaKaRa) and then transformed into E.coli DH5α competent cells for Sanger sequencing.