Lecture2mcb160L+f11

Lecture2mcb160L+f11 - Cross
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 Females

w
p[w+
Gal4];
+
;
+
;
+




x





males


w
;
+
;
TMS,
Sb
p[



2‐3]
;
+
 
















w
p[w+Gal
4]

+


+


+


























Y



+














LVM












+
 Females

w
p[w+
Gal4];

+
;
TMS,
Sb
p[



2‐3]






or




LVM

;



+
 


























w












+












+




































+









+
 Males





w
p[w+
Gal4];
+
;
TMS,
Sb
p[



2‐3]







or




LVM

;



+


 
























Y














+














+



































+









+
 Cross
1.1
 Control
cross

 Females




y


w


;

+
;
+
;
+






x




males
 Experimental
cross
 (1.1f)
 


















y



w





+


+


+

 (1.1a‐e)

 See
if

you
can
predict
the
expected
genotypes/phenotypes
if:

(1)
no
 transposiPon
occurs
(Mendelian
segregaPon)
or
(2)
if
the
P‐element
 transposes
to
an
autosome
in
the
male
germline
 Serial
DiluPons
 Conc.
stock
 culture
 20
ul
 mix
 1/100
 mix
 20
ul
 mix
 20
ul
 1/100
 1/100
 2
ml
 2
ml
 Tube
1
 DiluPon
factor
 relaPve
to
 original
stock
 2
ml
 Tube
2
 Tube
3
 ‐2
 0.02
ml
=
10
 
2ml
 ‐4
 10
 1/100
 1/100

x

 /100
 1 Plate
100
ul
 Plate
100
ul
 ‐6
 10
 1/100

x

 1/100


x
 
1/100
 Plate
100
ul
 ‐6
 (10




)
(
conc
original)
=
conc
Tube
3
 DNA
gel
electrophoresis
 ‐‐
 h\p://a32.lehman.cuny.edu/molbio_course/gelel.GIF
 +
 DNA
has
constant
 charge
to
mass
raPo,
 therefore
rate
of
 movement
is
 determined
by
 fragment
size…small
 fragments
move
more
 easily
through
the
gel
 matrix
 AnimaPon
of
gel
electrophoresis
at
 h\p://www.dnalc.org/ddnalc/resources/electrophoresis.html
 Generate
a
standard
curve
by
plo_ng
fragment
size
(y‐axis)
vs.

fragment
mobility
 100 kb 0.5% gel 10-30kb 10 kb 1% gel 0.5-10kb 1 kb 2% gel 0.1-1kb Log Length 0.1 kb Distance migrated The range of the DNA fragment sizes in the linear region of the plot depends upon the percentage of agarose in the gel. Bacterial Chromosome Plasmid DNAs Plasmid DNA is in Supercoiled form when purified from cells Plasmid
topoisomers
show
different
mobility
through
a
gel
 Nicked
circle
 Linear
fragment
 Supercoiled
plasmid
 Topoisomers
have
the
same
size
but
 different
conformaPon
 RestricPon
enzymes
recognize
and
cut
specific
nucleoPde
sequences
 (a common feature, but not always present) AnimaPon
on
restricPon
enzymes
can
be
viewed
at
 h\p://www.dnalc.org/ddnalc/resources/restricPon.html
 Star
Ac(vity
 Loss of specificity in a restriction enzyme digest is referred to as Star Activity. e.g. Eco RI recognizes and cuts the sequence GAATTC. Star activity of EcoRI is due to aberrant cutting at the sequence AATT, which occurs 16 times more often. In the gel on the left, Digest# 1 gave the expected pattern of bands Digests 2 and 3 gave additional smaller bands due to Star activity of EcoRI M 12 3 Star Activity can arise from a number of causes: 1.  Too much glycerol: Final glycerol conc. should never exceed 5% in a digest. Remember that the enyzme stock is in 50% glycerol. 2.  Low ionic strength: It is important to use the appropriate concentration of NaCl or other salt recommended for a particular enzyme. 3.  Too much enzyme: Do not overdigest - the amount of enzyme should be appropriate for the amount of DNA being digested. 4.  Presence of organic solvents like ethanol, DMSO etc: If solvents have been used to treat DNA, they can be removed by drying and re-suspending the DNA in aqueous solution Lambda
DNA
restricPon
digests
 EcoRI
–
HindIII
 double
digest
 HindIII
digest
 Note
that
EtBr
intensity
is
proporPonal
to
mass
of
fragments….in
 the
ladder
of
bands
each
fragment
is
present
in
equimolar
raPo
 but
larger
fragments
fluoresce
more
brightly.
Also
note
that
 bands
that
are
very
similar
in
size
may
not
be
resolved
and
may
 run
as
a
“doublet”
…the
unusually
bright
intensity
relaPve
to
size
 indicates

the
presence
of
twice
the
expected
mass
(two
 fragments
of
the
same

size)

 ...
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