Animal Cell Biotechnology: Methods and Protocols by Nigel Jenkins

By Nigel Jenkins

Eli Lilly & Co., Indianapolis, IN. experiences fresh learn and covers new suggestions utilized in genetically manipulating animal cells, optimizing their development in outlined media, warding off infection, and in harvesting and reading of mobilephone items. For researchers.

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DNA 500 ng/$) 1 0 pL, to 500 ng 1 Tfl polymerase (1 0 U/pL): 5 0 pL, to 5 U J H,O bidest 9 5 pL (ad 50 $ total volume) 2 Nick translation mix a 10x Nick Translation (GIBCO-BRL) buffer. 50 pL. 5 mA4 each dNTP (A, C, G): 1 2 pL, to 60 ~IJVf c c. c. d 10 mMCy3-dUTP. 0 pL, to 2 u + 200 pg g H,O bidest 11 8 pL (ad 50 pL total volume) The mck translation mrx is incubated for 4 h at 16”C, and the reaction is stopped by addition of 5 pL 200 mA4 EDTA. See also Note 2. 3. 7. Nuorescence In Situ Hybridization See also Notes 4-12.

5. 002 A4 EDTA), using a Bio-Rad Chef DR III apparatus, Blo-Rad, Hercules, CA. 5-s pulses over 16 h at 14°C. A smear or a ladder of discrete bands should be visible after ethldlum bromide staining of the gel, extending up to at least 200 kb (Fig. 3) 4. Notes 1 Results of m vitro amphficatlon. In vitro amphlicatlon has been used to express various genes m different cell lines (5,6,8) as summarized in Table 1 By comparing results with those from parallel transfectrons, m which nonllgated DNA of exactly the same composltlon had been employed, we observed that maxlmal production levels m clones from the m vitro amplification protocol were higher or as high as those from the control cotransfectlon, employing physically dlstmct DNA molecules.

The slides are then hybridized overnight in a humidified chamber at 37°C. 3 1 3 3 FLUORESCENCE SIGNAL DETECTION Slides hybridized with probes directly labeled with Cy3TM are washed three times m 60% formamlde/2x SSC at 45°C for 5 mm each and then rinsed in PN buffer. 5 pg/mL The fluorescence signals can be vlsuahzed m the metaphase preparations using a bandpassfilter (Omega,Brattleboro, VT; Chroma, Brattleboro, VT) for simultaneous vlsuallzatlon of DAPI (blue) and CY~~M (red) fluorescent signals.

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