Cloning. Cloning is what happens when a mentor, instead of listening and coaching the mentee, tries to shape the mentee in such a way that he or she becomes an exact replica of the mentor. The goal for a mentor is to advise, inform and support the choices of the mentee.
Cloning. The open reading frames of S-AHNAK and L-AHNAK N-terminus were amplified from human and mouse skeletal muscle cDNA and cloned into pET28 (prokaryotic) and pSG8 (eukaryotic) expression vectors with unique restriction sites for BamHI and XhoI. In addition mouse S-AHNAK was cloned into pIVEX1.3 for in vitro transcription-translation. Exon-exon boundary spanning amplicons were separated on agarose gels, purified and cloned in pTOPO-blunt vector (Invitrogen). All vector cloned sequences were confirmed by direct sequencing (LGTC, Leiden, the Netherlands). Transfection Cells were plated and grown to 50% subconfluence. U2OS cells were transfected with FuGENE-6 (Roche Applied Science) and C2C12, IM2 and immortalized myoblasts were transfected with Lipofectamine PLUS (Invitrogen), both according to manufacturers protocol. 24h Post-transfection cells were washed and harvested for further analysis. Recombinant protein production pET28 expression constructs were transformed into BL21 (DE3)-RIL Escherichia coli (Stratagene) cells by electroporation. Bacterial cells were grown to log phase and recombinant protein production was initiated by the addition of IPTG (Fermentas, Germany) to a final concentration of 1 mM. After 2.5 h induction at 37ºC, the cells were collected by centrifugation at 3,000 g, and lysed in lysis buffer 1 (50 mM Tris, pH 7.4, 1 mM EDTA, 1.5mg/ml lysozyme, 0.15 M NaCl, 1% Triton X-100, plus 1× protease inhibitor cocktail (Roche Molecular Biochemicals, Basel, Switzerland)). All proteins were in the resulting soluble fraction except AHNAK N-terminus, which was extracted by 5 min sonication on ice. Lysates were spun down at 13,000 g for 30 min at 4ºC, and the proteins purified via IMAC with a Profinia purificator (BioRad). Concentration was obtained with absorption spectroscopy, and protein stability analyzed on SDS-PAGE gels. An in vitro translation was performed with S-AHNAK in pIVEX vector using the wheat-germ in vitro transcription translation kit according to the manufacturer’s protocol. HIS6-tagged proteins were subsequently purified using TALON resin (Clontech). In short TALON resin was pre-equilibrated with PBS and added to the protein mixture. Samples were incubated for 2h, 4°C, and subsequently spun down. After 3 wash steps (5 min) the TALON resin was loaded onto a column and subjected to a 300x bed volume wash. Proteins were eluted with 200mM imidazole, and dialyzed O/N at 4°C to PBS. Concentration and purity were analyzed on SDS- PAGE gels. ...
Cloning. Long ago, in Old Reckoning timeç before The Computer began guiding Alpha Complex— before Alpha Complex even exiçted!—humanç once reproduced by rutting, like vile animalç. Xxxxxxxx and expreççionç of revulçion are permitted, though not mandatory. Xxxxxxx The Computer careç deeply for all citizenç, it now growç them in clone tankç. Thiç iç much leçç meççy and diçguçting than the old way, and far leçç random. The Computer ençureç every human citizen iç genetically perfect in all reçpectç. Improved by a program of mandatory hormone çuppreççantç, human citizenç are no longer troubled by the beçtial urgeç of paçt generationç. The Computer'ç reçearcherç in R&D continue to pioneer çtunning advanceç in cloning *UNIOR #ITIZENS IN !LPHA #OMPLEX NEED NOT SUFFER THE INDIGNITIES OF HISTORICAL CHILDBIRTH. 42534 4(% #/-054%2! 4(% #/-054%2 )3 9/52 &2)%.$! 13 0,!9%2 3%#4)/. #,%!2!.#% 2%$ Serve The Computer well, and you too may join [NAME DELETED]! The Computer alço attendç regretfully to the pervaçive preçence of traitorç. Traitorç are everywhere! Though final cruçhing victory over Communiçt çubverçion iç cloçe at hand, the traitorç' laçt çhrill treaçon-laden gaçpç çtill endanger their innocent victimç. Fight on, Troubleçhooter, fight on for The Computer and all Alpha Complex! ® Locate and apprehend or deçtroy unregistered mutants. They endanger human genetic purity and muçt be terminated or enliçted in our great cauçe. Do not terminate registered mutants, innocent citizenç who realized they poççeçç, through no fault of their own, a treaçonouç mutant power. Theçe unwitting traitorç promptly reported their power to The Computer. In itç wiçdom and benevolence, The Computer forgiveç thoçe who make full confeççion, and it permitç regiçtered mutantç to live. However, The Computer requireç regiçtered mutantç to wear a uniform with yellow çtripeç at all timeç. (YELLOW Clearance mutantç wear a black- bordered yellow çtripe.) Regiçtered mutantç are not çubject to apprehençion or termination when they uçe their powerç. Encourage theçe former traitorç to uçe their inhuman powerç reçponçibly, for the good of all Alpha Complex, ço they may çomeday overcome their unfortunate terrible çtigma. ® Locate and apprehend or deçtroy members of secret societies. They are traitorç, often terroriçtç, and muçt be brought to juçtice. The exiçtence of many çecret çocietieç iç public knowledge. It iç not technically illegal to diçcuçç their treaçonouç doctrineç and objectiveç, the better to enforce go...
