Method for quickly constructing siRNA carrier by using pSilencer plasmid
A carrier and fast technology, applied in the direction of using carrier to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of long fragments and high cost of siRNA, and achieve the effect of reducing cost, reducing the probability of error, and saving construction time
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[0041] 1. Synthesis of siRNA gene fragments and PCR completion reaction:
[0042] 1) According to the sequence of the bc1212 gene, find the target of the interfering gene (GCTGGGCATCAGTGAGGAC), and synthesize the upper and lower gene sequences according to the instructions of pSilencer plasmid construction. Including adding a circular base sequence in the middle of the sequence, adding HindIII and BamHI enzyme cutting sites upstream and downstream, and adding protective bases at both ends of the enzyme cutting site to facilitate subsequent enzyme cutting (by Shanghai Boya company synthesis).
[0043] Upstream gene sequence (53bp):
[0044] 5'-GCGGATCCGCTGGGCATCAGTGAGGACATTCAAGAGATGTCCTCACTGATGCC-3'
[0045] Downstream gene sequence (53bp):
[0046]5'-GCAAGCTTTTCCAAAAACTGGGCATCAGTGAGGACATCTCTTGAATGTCCTC-3'
[0047] 2) PCR reaction to complete the siRNA gene fragment:
[0048] Pyrobest DNA Polymerase (purchased from Takara): 2.5U / 0.5ul
[0049] Upstream gene sequence: 5ug ...
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