Experimentalcross
(1.1f)
yw+++
(1.1ae)
Seeifyoucanpredicttheexpectedgenotypes/phenotypesif:(1)no
transposiPonoccurs(MendeliansegregaPon)or(2)ifthePelement
transposestoanautosomeinthemalegermline
Seri;+xmales Cross1.0
Femaleswp[w+Gal4];+;+;+xmalesw;+;TMS,Sbp[23];+
wp[w+Gal4]+++Y+LVM+
Femaleswp[w+Gal4];+;TMS,Sbp[23]orLVM;+
w++++
Maleswp[w+Gal4];+;TMS,Sbp[23]orLVM;+
Y++++
Cross1.1
Controlcross
Femalesyw;+;+alDiluPons
Conc.stock
culture
20ul
mix
1/100
mix
20ul
mix
20ul
1/100
1/100
2ml
2ml
Tube1
DiluPonfactor
relaPveto
originalstock
2ml
Tube2
Tube3
2
0.02ml=10
2ml
4
10
1/100
1/100x /100
1
Plate100ul
Plate100ul
6
10
1/100x
1/100x
1/100
Plate100ul
6
(10)(concoriginal)=concTube3
DNAgelelectrophoresis
h\p://a32.lehman.cuny.edu/molbio_course/gelel.GIF
+
DNAhasconstant
chargetomassraPo,
thereforerateof
movementis
determinedby
fragmentsizesmall
fragmentsmovemore
easilythroughthegel
matrix
AnimaPonofgelelectrophoresisat
h\p://www.dnalc.org/ddnalc/resources/electrophoresis.html
Generateastandardcurvebyplo_ngfragmentsize(yaxis)vs.fragmentmobility
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
Plasmidtopoisomersshowdierentmobilitythroughagel
Nickedcircle
Linearfragment
Supercoiledplasmid
Topoisomershavethesamesizebut
dierentconformaPon
RestricPonenzymesrecognizeandcutspecicnucleoPdesequences
(a feature, common but not always
present)
AnimaPononrestricPonenzymescanbeviewedat
h\p://www.dnalc.org/ddnalc/resources/restricPon.html
StarAc(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
LambdaDNArestricPondigests
EcoRIHindIII
doubledigest
HindIIIdigest
NotethatEtBrintensityisproporPonaltomassoffragments.in
theladderofbandseachfragmentispresentinequimolarraPo
butlargerfragmentsuorescemorebrightly.Alsonotethat
bandsthatareverysimilarinsizemaynotberesolvedandmay
runasadoublettheunusuallybrightintensityrelaPvetosize
indicatesthepresenceoftwicetheexpectedmass(two
fragmentsofthesamesize)