In this study, a method to remove residual powder on a multi-layered graphene and a new approach to transfer multi-layered graphene at once are studied. A graphene one-step transfer (GOST) method is conducted to minimize the residual powder comparison with a layer-by-layer transfer. Furthermore, a residual powder removing process is investigated to remove residual powder at the top of a multi-layered graphene. After residual powder is removed, the sheet resistance of graphene is decreased from 393 to 340 Ohm/sq in a four-layered graphene. In addition, transmittance slightly increases after residual powder is removed from the top of the multi-layered graphene. Optical and atomic-force microscopy images are used to analyze the graphene surface, and the Ra value is reduced from 5.2 to 3.7 nm following residual powder removal. Therefore, GOST and residual powder removal resolve the limited application of graphene electrodes due to residual powder.

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures〔10% (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.〕which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into LN2. One-step dilution in straw was done in 25℃ water for 1 min, by placing vertically in the state of plugged- end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.