Sentence examples for and subsequently inserting the from inspiring English sources
PMMA reinforcement of pedicle screws can be carried out by first injecting the cement into the pedicle and subsequently inserting the screw.
Then the homologous recombination vector pPuKAd was obtained by subsequently inserting the ack-down between the BamH I and Not I sites of pBlue- pta- aph.
Then, the Kan was digested with the nuclease SalI and subsequently inserted into the recombinant plasmid pMD19-Up-Down to form the plasmid pMD19-Up-Kan-Down.
The COX-2 (-327/+59) promoter fragment [ 42] was PCR amplified with the primers 5'- GandTCGAAGAAGAAAAGACATCTGGCG-3' and 5'- CTCGAGGCGCTGCTGAGGAGTTCCTG-3' and subsequently inserted into the Sac I/ Xho I sites (underlined in the primer sequence) of the luciferase reporter vector pGL3-Basic (Promega, Madison, WI).
Fragments of the heterologous promoters, except CaMV 35S long), were excised with SalI and XhoI and subsequently inserted between the SalI and XhoI sites of the three basic vectors pluc, pluc-Ω and pluc-enh, respectively.
To make Plac- efe-Flag plasmid, the efe-Flag ORF from pJU105 was PCR amplified, digested with HindIII and KpnI and subsequently inserted into the pUC-based pGFPuv vector digested with the same enzymes.
Fragments of sGFP and sGFP-3′-UTR were amplified from pTKM1-sGFP-3′-UTR plasmid and cloned into pENTR/D-TOPO (Invitrogen) and subsequently inserted into the pT1OG vector (LC126301)40 (Supplementary Fig. 4a,b).
For complementation of cas8, pTA927-N-FLAG- cas6 [ 22] was digested with HindIII and BamHI to remove cas6 and to subsequently insert the cas8 gene.
The resulting fragment was recombined into pDONR207 as described above and subsequently inserted into pEARLEYGATE302 using the Gateway LR clonase reaction (Invitrogen ®).
Then the ICP34.5 US and DS FLRs were jointed together using an overlapping PCR with the primer pair ICP34.5USf/ICP34.5DSr and subsequently inserted into pSP72 (Promega) pre-digested with BamHI/ XhoI and treated with T4 DNA polymerase for blunt-end cloning.
Resulting PCR product was digested using XhoI and BamHI restriction enzymes, and subsequently inserted into pCDNA3-bestrophin-1 plasmid digested with XhoI/BamHI.
