Barnea dilatata mitochondrial COI gene amplification primer
A wide-shelled whole sea bamboo shoots, amplification primer technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods and other directions, can solve problems such as unsatisfactory amplification results, reduce the time and cost of amplification, Highly specific and efficient effects
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Embodiment 1
[0017] Example 1: Amplification and sequence analysis of the COI gene fragment of the whole sea bamboo shoot and several other shellfish genome COI gene fragments
[0018] Amplification of the COI gene of the wide-shelled whole sea bamboo shoot:
[0019] (1) DNA extraction was performed on wide-shelled whole sea bamboo shoots collected from Ningbo and Qinhuangdao, Zhejiang Province (16 in total, 32 in total) by CTAB method, and diluted with double distilled water to a working solution of 100 ng / μl for later use.
[0020] (2) Use the above primers to perform PCR amplification on the DNA working solution. The preparation solution of the reaction system includes: 1 μl template DNA, 0.05 μl Taq polymerase, 1 μl 10×PCRb μffer, 0.8 μl NTPs, 1 μl each of the upstream and downstream primers, 5.15 μl double-distilled water. The PCR amplification conditions were: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 45 s, annealing temperature at 46-56°C for 45 s, extension at 7...
Embodiment 2
[0023] Embodiment 2: the design of primer
[0024] (1) DNA extraction was performed on wide-shelled whole sea bamboo shoots collected from Ningbo and Qinhuangdao, Zhejiang Province (16 in total, 32 in total) by CTAB method, and diluted with double distilled water to a working solution of 100 ng / μl for later use.
[0025] (2) Using COI universal primers (LCO1490F and HCO2198R, Folmer1994) to amplify the partial sequence of the whole sea bamboo shoot with broad shell.
[0026] (3) From the part of the wide-shell whole sea bamboo sequence obtained above, use the software BioEdit (http: / / www.Mbio.Ncsu.edu / BioEdit / bioedit.html) to perform multiple comparative analysis on the above sequence to determine the sequence before and after Conserved area.
[0027] (4) After comparison, 150 base pairs at the front and rear ends are in the conserved region, and 3 pairs of primers were designed with the primer design software PrimerPremier5. The primers designed were as far as possible witho...
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