In our initial study, we tried to optimize the model procedure mentioned above by detecting the efficiency of different reaction conditions, such as piperidine/EtOH, p-TSA/EtOH, AcOH/EtOH, AcONa/AcOH, H2SO4/AcOH, DBSNa/MeOH, DBSNa/EtOH, DBSNa/H2O, DBSNa/AcOH (Table 2).
The red star indicated the conversion of C to T. To detect the efficiency of base editing, the region around the target sites was amplified and analyzed initially by the T7EN1 cleavage assay.
To detect the efficiency of bacterial escape from the phagosome, cells infected with the L. monocytogenes strain NF-L327 were spun and fixed as previously described.
We used high-resolution electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry to confirm protein primary structures and to detect the efficiency of coupling maleimide into Avd(S16C).
PCR primers used to detect the efficiency of recombination of Fbw7 and R26 alleles are given in the Supplemental Experimental Procedures.
Moreover, pEGFP plasmid was transfected in equal amounts in both of the previous transfections in order to detect the efficiency of DNA intake (Additional file 1: Figure S1).
Afterwards, a qRT-PCR with a dilution series was performed to detect the efficiency of the qRT-PCR reaction and to prove that only one product can be detected within the melting curve.
In addition, individuals from one population of B. asiatica and one population of B. acutangula were also sampled to detect the efficiency of these markers in cross-species amplification (Appendix 1).
In order to detect the efficiency of transfection in Fadu cells, 2 × 10 Fadu cells were seeded into 15 mm plate specially used for laser scanning confocal microscope and incubated for 24 h.
In addition, individuals from one population of C. pectinifolia (Heishiding Natural Reserve, Fengkai, Guangdong; 23°31′12″N, 111°52′13″E; Y. Liu 090718) and one population of C. garciniifolia (Xishuangbanna, Yunnan; 21°55′50″N, 101°15′09″E; Y. Huang 140211) were also sampled to detect the efficiency of these markers in cross-species amplification.
Thirty-six detection points (the distance toward the direction of the extended line is 8.4 mm, and that toward the peripheral direction is 10°) were set in one helical lead with the purpose of detecting the correction efficiency of this polishing method with regard to errors in shape.
