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.
Resulting PCR product was digested using XhoI and BamHI restriction enzymes, and subsequently inserted into pCDNA3-bestrophin-1 plasmid digested with XhoI/BamHI.
T6SS genes were amplified using indicated primers (see Appendix), cloned into plasmid pJet (Thermo Scientific) and subsequently inserted into pBAD24.
The resulting fragment was recombined into pDONR207 as described above and subsequently inserted into pEARLEYGATE302 using the Gateway LR clonase reaction (Invitrogen ®).
A 1977 bp D6PK promoter-PCR fragment (D6PKp) was cloned into pCR2.1 (LifeTechnologies, Carlsbad, CA) and subsequently inserted as KpnI/ XhoI fragment into pGREEN-YFP D6PK pGREEN-YFP D6PKKp:YFP:D6PK.
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.
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.
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.
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).
How do tandem repeats form, and subsequently insert and delete in mt genomes, and why do they have high sequence and length heteroplasmy even within an individual?
