7717

• UCREFRP Catalog Number: 7717
• Author: Rosenfeld, M. J.
• Title: Genetic Analysis of the Colorado River Gila
• USFW Year: n.d.
• USFW - Doc Type: Non-fatal Sampling, Cell Culture, Chromosomal Analysis and Electrophoresis.
• Copyright Material: NO

order

to ml/ll/lIlZt:' CI:O'J.l

UC'III'-'\~'-.:'

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replanted into a 75 cm2 flask (Corning).

When cells covered the vessel surface

(= confluency), e~ch

secondary culture was trypsinized and divided into two 75 cm2

flasks, one. for 'cytogenetics' and the other for frozen stor~ge

of

live cells.

Usually the 'cytogenetics' flask was grown, to

confluency and split into 2 or 3 flasks.

Chromosome spreads w~re

harvested when cells covered 2/3-3/4 of the vessel surfac.s.

About 4 h before cell

harvest,

dimecolcine

(Sigma)

was ad~led

to a final concentration of 0.8 ug/ml.

The harvesting of c~lls

followed Amemiya et a1. (1984) with 2 e~:ceptions.

CuI tures WE.-re

trypsinized as described above and centrifugations were don~ at

,.

100 X g at 40 C for 7 min.

Cell

suspensions were

-. --':~
fixed 1m at

. 'I

',. - :'~ :...~ -:

I!"f' .

. ..'.<'i .
~;t

~'....' -": ~-

. ~, <. ;~t~:;~-'1~h.~:~ .~. .

least 3 changes of 3: 1 absolute methanol - acetic acid .at~JJ~' C,. '~

,,'f::'~~f~\{f

Slides were cleaned with tissue culture detergent, plac~d in

each for 15 min.

~

3X distilled

H20 and

kept in

the refrigerator

for a~ leas~~ 4h

prior to use.

Finished preparations were made by' depositing!, 1-3

drops of

cell suspension onto wet chilled slides with chrom~some

complements spread by breathing

onto the slide surface

I
(SL\n et

aI., 1974).

After drying at room temRerature~ slides were

ored

in a dust-free container.

Gross staining was done in 4% Giemsa (Fisher;

obtai ned .as a

l.' ;... l

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j c.. .-

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