Praktikumblock
WS 2005/2006
Evolutionary Developmental Biology,
07.11.2004 – 18.11.2004
Kursraum C
Institute für Genetik
Zülpicherstraße 47
Wim Damen
Institut für Genetik
Abteilung für Evolutionsgenetik
Zülpicher Straße 47
Universität zu Köln
Tel 0221 470 3419
damen@uni-koeln.de
Raum 1.01
Wim Damen
Evelyn Schwager
Maarten Hilbrant
1
Gruppe C
20 Henrich
21 Hopfen
13 Elfimova
54 Thorn
40 Nickels
47 Potting
16 Gürtler
35 Meinecke
10 Dip
17 Häming
8 Campos da Ponte
9 Charara
28 Kümmler
52 Stermann
Alexander
Corinna
Natalia
Andreas
Jochen
Christoph
Anne
Anke
Phat Vinh
Daniela
Julia
Nadia
Nils Christian
Jacek
C1
C1
C2
C2
C3
C3
C4
C4
C5
C5
C6
C6
C7
C7
2
Schedule:
Montag 07.11.
0
Introduction
1
Sequence alignments and primer design for PCR
2a
Start PCR reactions: Primary PCR
14/15 Fixation Drosophila and Tribolium embryos
Dienstag 08.11.
2b
Continuation of PCRs: Nested PCR
3
Preparation of agarose gel and analysing PCRs on agarose gel
4a
Prepare preparative agarose gel (for tomorrow morning)
14/15 Fixation Drosophila and Tribolium embryos
Mittwoch 09.11.
4b
Run preparative agarose gel
5
isolation of PCR-Fragments (Minelute)
6
Cloning into plasmid vector (TOPO kit)
7
Transformation to bacteria
14/15 Fixation Drosophila and Tribolium embryos
Donnerstag 10.11.
8
Analysis of transformation by colony PCR
9
Inoculation of cultures
13
Preparing DIG labeled probes
14/15 Fixation Drosophila and Tribolium embryos
Freitag 11.11.
10
Isolation of plasmid DNA
11
Cycle sequencing
14/15 Fixation Drosophila and Tribolium embryos
Montag 14.11.
12
Sequence analysis
16/17 Starting the in situ hybridisation for the fruit fly Drosophila, the beetle
Tribolium, and the spider Cupiennius
Dienstag 15.11.
12
Sequence analysis
16/17 Continuation of the in situ hybridization
16/17 Staining for the Tribolium and Drosophila in situ
Mittwoch 16.11.
12
Sequence analysis
18
Analysis of the embryos (make drawings)
16/17 Staining for the Cupiennius in situ
Donnerstag 17.11.
12
Sequence analysis
18
Analysis of the embryos (make drawings)
Freitag 18.11.
19
Wrap up: Student presentations and general discussion of the results
3
Evolutionsgenetik: Evolution entwicklungsbiologischer Prozesse
Wim Damen, Evolutionsgenetik, Institut für Genetik Weyertal 121
Tel 470 3419
e-mail: damen@uni-koeln.de
Evolutionary Developmental Biology
Evolutionary Developmental Biology tries to reveal the underlying similarities
and differences between species by looking at the genes that control
developmental processes. Goal of the experiments is to get an impression of
the evolution of developmental mechanisms and the evolution of body plans
in arthropods. During the course, an important class of developmental genes,
the segmentation genes, will be studied in different arthropod species.
Segmentation genes control the formation of the segments, repeated units of
the arthropod body plan, and have been studied in detail in the fruit fly
Drosophila (insects). We will isolate segmentation gene homologues in a PCR
approach from other arthropods (spider, pycnogonids, brine shrimp, millipede,
others?). The PCR fragments that will be obtained from the different species
will be cloned, sequenced, and analysed. The next step then is to analyse the
expression patterns of segmentation genes and compare them with each
other and with the expression patterns of homologous of these genes known
from other species. For these expression studies, we will use embryos of the
fly Drosophila melanogaster, the beetle Tribolium castaneum, and the spider
Cupiennius salei. These comparisons may allow us to draw conclusions on the
evolution of the segmented arthropod body plan.
SELECTED REFERENCES:
Damen, W.G.M., Hausdorf, M., Seyfarth, E.-A. and Tautz, D (1998) A conserved mode of head
segmentation in arthropods revealed by the expression pattern of Hox genes in a spider. Proc.
Natl. Acad. Sci USA 95, 10665-10670
Damen, W.G.M., Weller, M. and Tautz, D. (2000) The expression patterns of hairy, even-skipped,
and runt in the spider Cupiennius salei imply that these genes were segmentation genes in a
basal arthropod. Proc. Natl. Acad. Sci USA 97,4515-4519
Davis, G.K. and Patel N.H. (1999) The origin and evolution of segmentation. Trends Genet. 15,
M68-M72.
Damen WGM (2002) Parasegmental organization of the spider embryo implies that the
parasegment is an evolutionary conserved entity in arthropod embryogenesis. Development 129,
1239-1250
Stollewerk, A., Schoppmeier, M. and Damen, W.G.M. (2003) Involvement of Notch and Delta
genes in spider segmentation. Nature 423, 863–865.
Peel, A. and Akam, M., 2003. Evolution of segmentation: rolling back the clock. Curr. Biol. 13,
R708–R710.
Tautz D (2004) Segmentation. Dev. Cell 7, 301-312.
4
Praktikumsblock “Evolutionary Developmental Biology”
During this course we will do two major experiments. First, we will do a PCR
experiment and amplify gene fragments of segmentation genes (e.g. Hox
genes) by PCR. These PCR fragments will be cloned and sequenced. The
different gene fragments found will be compared among the different
arthropod species, using data known from Genbank. We will clone Hox gene
fragments from various arthropod species (depending on the availability we
will use DNA from a seaspider (pycnogonids), a spider (Tegenaria), the brine
shrimp Artemia franciscana, and others??).
In a parallel experiment, we will do expression studies via in situ
hybridisations for Hox genes and other segmentation genes in the spider
Cupiennius salei, the beetle Tribolium castaneum, and the fruit fly Drosophila
melanogaster and compare the expression patterns with the ones known from
the fly Drosophila. Similarities and differences among them may tell us
something about the evolution of developmental processes and animal body
plans.
Amplification of segmentation genes by PCR
Our aim is to isolate via PCR fragments of Hox genes from different arthropod
species.
Flow chart:
(1) Make sequence alignment and design of primers directed against
conserved parts in the proteins
(2) PCR reactions, using the primers we designed in step (1)
(3) Cloning of the PCR fragments that we (hopefully) will obtain
(4) Sequencing of the cloned PCR fragments.
(5) Analyses and comparisons of the different sequences obtained.
(6) Discuss and compare with results of other groups
Hox gene expression analysis via in situ hybridisation
Our aim is to analyse the expression patterns for the different Hox genes and
compare them.
Flow chart:
(1) Fix Tribolium and Drosophila embryos
(2) Prepare DIG labelled probes
(3) Prepare (devitelinize) embryos of Cupiennius
(3) Perform in situ hybridisation
(4) Analysis of expression patterns
(5) Make drawings of embryos and comparison of data for the different
Hox genes in the different animals
(6) Discuss and compare with results of other groups
5
1 Design of primers.
We like to isolate and clone Hox gene fragments from various arthropods. The
Hox proteins of the fly Drosophila as well as the ones of vertebrates contain a
conserved domain. This so-called homeodomain is the DNA binding domain of
the protein. An alignment of the homeodomains of the Drosophila Hox genes
is depicted below. As these domains are highly conserved at the protein level
between these eight Drosophila Hox genes but also between the Hox genes of
Drosophila and the Hox genes of vertebrates, we assume that these domains
are also conserved in other animals. If we design primers against these
domains (via reverse translation, remember the wobbles!), we can use these
primers in a PCR reaction on either cDNA or genomic DNA to isolate Hox gene
fragments from these arthropods.
Alignment of Drosophila Hox genes:
Dm-lab
Dm-pb
Dm-Dfd
Dm-Scr
Dm-Antp
Dm-Ubx
Dm-abd-A
Dm-Abd-B
Hexapeptide
homoeodomain
IPTYKWMQLK<##>NNSGRTNFTNKQLTELEKEFHFNRYLTRARRIEIANTLQLNETQVKIWFQNRRMKQKKRV
VPEYPWMKEK<##>PRRLRTAYTNTQLLELEKEFHFNKYLCRPRRIEIAASLDLTERQVKVWFQNRRMKHKRQT
RIIYPWMKKI<##>PKRQRTAYTRHQILELEKEFHYNRYLTRRRRIEIAHTLVLSERQIKIWFQNRRMKWKKDN
PQIYPWMKRV<##>TKRQRTSYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEH
SPLYPWMRSQ<##>RKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEN
HTFYPWMAIA<##>RRRGRQTYTRYQTLELEKEFHTNHYLTRRRRIEMAHALCLTERQIKIWFQNRRMKLKKEI
LPRYPWMTLT<##>RRRGRQTYTRFQTLELEKEFHFNHYLTRRRRIEIAHALCLTERQIKIWFQNRRMKLKKEL
PGLHEWTGQV<##>VRKKRKPYSKFQTLELEKEFLFNAYVSKQKRWELARNLQLTERQVKIWFQNRRMKNKKNS
6
2 PCRs
During this course we try to amplify Hox genes of different arthropods. We
use either cDNA prepared from embryonic RNA or genomic DNA as templates
in the PCR reaction. The cDNA is made from embryonic RNA using either an
oligo-dT primer or random primers in a reverse transcription reaction.
Use the primers as designed in ‘1’. The PCR conditions depend on the primers
used!
Don’t forget to do a no-template control (use water instead of cDNA)
Design PCR reaction, and discuss with course assistants.
Please use filter-tips when you pipet your PCR reaction!!
2a
2
3
3
1.5
1.5
19
0.3
30
Initial PCR
µl template (cDNA or genomic DNA)
µl 10 x buffer
µl dNTPs (2mM stock)
µl primer-1 (20 µM stock)
µl primer-2 (20 µM stock)
µl water
µl Taq polymerase (1.5 unit)
µl total
Cycle:
1 min 94°C
35x
30 sec 94°C
2 min XX°C
1 min 72°C (if expected gene fragment is larger than 1 kb, use 1 min/kb)
5 min 72°C
4°C
forever
2b
2
3
3
1.5
1.5
19
0.3
30
Nested PCR
µl initial PCR reaction
µl 10 x buffer
µl dNTPs (2mM stock)
µl primer-3 (20 µM stock)
µl primer-4 (20 µM stock)
µl water
µl Taq polymerase (1.5 unit)
µl total
Cycle:
1 min 94°C
35x
30 sec 94°C
2 min XX°C
1 min 72°C (if expected gene fragment is larger than 1 kb, use 1 min/kb)
5 min 72°C
4°C
forever
7
3 Analytical agarose gel
To test whether we amplified a DNA fragment of the right size (what is the
right size?) in our PCR, we will analyse a small aliquot of the PCR reaction on
an agarose gel. Prepare agarose gel (1.5%).
Analyse 3 µl of the initial PCR reactions and 3 µl of the nested PCR reactions
on the agarose gel. Don’t forget the control reaction and the size standard!
Isolation and cloning of PCR fragments
If we get bands of the expected size in our gel in ‘3’, we would like to clone
these PCR products into a cloning vector. Discuss with the course assistants
which fragment has the right size and which one you like to clone. It depends
on the vector and cloning strategy, whether we have to make the DNAfragments blunt-ended and whether we have to phosphorylate the DNA
fragment. We plan to use a TOPO-TA cloning kit (Invitrogen). This kit does
not require phosphorylation and polishing of the ends. To remove all primers,
nucleotides, and buffer components from the PCR fragments, and to ensure
that we only clone the fragment of the right size, we separate the fragments
on a gel ‘4’, cut the band of the expected size from the gel, and isolating the
DNA from the gel slice ‘5’.
Finally, we ligate the fragment into the TOPO cloning vector ‘6’ and transform
E. coli TOP10 cells with this construct ‘7’.
4 Running the PCR products on a preparative agarose gel
In order or get rid of the dNTPs, salt, etc, and to remove DNA fragments of
the wrong size, we purify the PCR products on an agarose gel before we ligate
them into the plasmid vector. To so do, prepare a 1.5 % gel (at least one
hour before you need the gel!). Add 5 µl of loading buffer (blue juice) to the
selected PCR reaction (everything that is left, is about 25 µl) and load onto
the agarose gel (with wide wells). Run at 80V (2-3 hours). Cut the band
containing your fragment from the gel, please cut the slice of gel as small as
possible. Isolate the DNA fragment from the gel slice using the Minelute kit
(Qiagen) or PureLink kit (Invitrogen) ‘5’.
8
5 DNA isolation from gel slice
5a MinElute Gel extraction kit (Qiagen).
• Excise the DNA fragment from the gel
• Weigh the gel slice
• Add 3 volumes of buffer QG to 1 volume gel slice (100 mg gel slice ~ 100
µl)
• Incubate at 50˚C for 10 min, until the gel slice has completely dissolved.
Mix every 2-3 min. After the gel slice has completely dissolved, the color of
the mixture must be yellow.
• Add one gel volume of 2-propanol, mix but do not centrifuge.
• Place a MinElute spin column in a 2-ml collection tube (kit)
• Apply the sample to the MinElute spin column und centrifuge for 1 min
• Discard flow through and place MinElute spin column back into the same
collection tube
• Add 500 µl QG buffer to the MinElute spin column and centrifuge for 1 min
• Discard flow through and place MinElute spin column back into the same
collection tube
• Add 750 µl PE buffer to the MinElute spin column and centrifuge for 1 min
• Discard flow through. Place MinElute spin column back into the same
collection tube
• Centrifuge for an additional min. at max speed.
• Place MinElute spin column back into clean 1.5 ml tube
• Add 10 µl of buffer EB to elute the DNA.
• Centrifuge for 1 min at max speed
• DNA is now in tube
5b PureLink Quick Gel extraction kit (Invitogen).
• Excise the DNA fragment from the gel
• Weigh the gel slice
• Add 3 volumes of buffer GS1 to 1 volume gel slice (100 mg gel slice ~ 100
µl)
• Incubate at 50˚C for 15 min, until the gel slice has completely dissolved.
Mix every 2-3 min.
• Afer the gel slice appears dissolved, incubate for an additional 5 min.
• Place a Quick Gel Extraction spin column in a 2-ml collection tube (kit)
• Load the sample onto the column und centrifuge for 1 min
• Discard flow through and place spin column back into the same collection
tube
• Add 500 µl GS1 buffer to the spin column, incubate at room temperature
for 1 min, and centrifuge for 1 min
• Discard flow through and place spin column back into the same collection
tube
• Add 700 µl W9 buffer to the spin column , incubate at room temperature
for 5 min, and centrifuge for 1 min
• Discard flow through. Place spin column back into the same collection tube
• Centrifuge for an additional min. at max speed to remove any residual
wash buffer W9.
• Place spin column back into clean 1.5 ml tube
• Add 30 µl of warm (70˚C) buffer EB to elute the DNA, incubate at room
temperature for 1 min
• Centrifuge for 2 min at max speed
• DNA is now in tube
9
6 Ligation of PCR fragments into pCR ®2.1-TOPO®.
• Add (in this order!):
4 µl fragment from ‘5’
1 µl salt solution
1 µl pCR®2.1-TOPO® vector
6 µl
• Incubate for 5 minutes at RT (22-23˚C)
• Place on ice until transformation
10
7 Transformation to TOP10 cells
Prepare plates:
Place bottles with LB agar in microwave and melt the agar. Let cool to about
50-60 ˚C. Add 1/1000 of the Amp stock solution to the agar (400 µl Amp per
400 ml medium). Pour plates (about 20-25 ml agar/plate), avoid air-bubbles
in the agar. Let the plates cool to RT. Once the plates are cooled, put 100 µl
of X-gal (2%) onto the plates and spread over the plate. Depending on the
kind of cells available, we use protocol 7a (electro-competent cells) or
protocol 7b (chemical competent cells).
7a Electrotransformation of TOP10 cells
• Place the electro-cuvet on ice and let it cool.
Be sure there is NZY+ medium
• Place the bacteria from the freezer directly on ice and thaw them one ice.
• Add 0.7 µl of the ligationreaction (P6) to the bacteria
• Pipet the bacteria between the plate of the electroporation cuvet
• Place in the correct way in the electroporator (BioRad), cuvet fits in only
one way
Setting are 1.8 kV
Press both PULSE buttons, After a few second the machine says “bleeeep”,
which means ‘I’m ready’.
• Add immediately 1 ml of NZY+ medium and than transfer the cells to a 15
ml tube.
• Incubate for 1 h in the shaker at 37˚C
• Plate 200 µl of the bacteria on an Amp-plate with X-gal and incubate at
37˚C (o/n)
7b Transformation to chemical competent TOP10 cells
• Be sure that:
waterbath is at 42˚C
SOC medium is thawed and is at RT
Plates with antibiotic are at 37˚C
• Thaw on ice one vial of One shot TOP10 cells
• Add 2 µl of the cloning reaction (P6), mix gently by tapping. Do not mix
by pipetting!!
• Incubate on ice for 20-30 min
• Heat-shock for exactly 30 sec at 42˚C
• Place tubes immediately on ice
• Add 250 µl of RT SOC medium
• cap tube tightly and shake at 37˚C for 1 hour
• Plate 50 µl of the transformation on a pre-warmed plate
Plate the remaining (~250 µl) of the transformation on a second prewarmed plate
• Place plate in 37˚C incubator
11
8 Colony PCR
After we obtained colonies on our plates, we would like to test the (white)
colonies for insert size to test whether the plasmid indeed contains an insert
of the expected size. (Why the white colonies and not the blue ones?). To do
so, we will perform a colony PCR on a number of colonies. For each tested
colony we will save the bacteria on a backup plate.
-Prepare 24 tubes with 10 µl each of:
1
1
0.5
0.5
7
0.02
µl
µl
µl
µl
µl
µl
10x PCR buffer
dNTPs
pBS-A oligo
pBS-E oligo
water
Taq polymerase (0.1 units)
Take a wooden toothpick, touch one of the white colonies on the petridish,
stick into one of the PCR tubes for about 1 second, and next, stick into the
backup Petri-dish with a numbered grid to save the bacteria.
PCR cycle
1 min 94°C
30x
30 sec 94°C
45 min 60°C
1 min 72°C
5 min 72°C
4°C
forever
Add 2 µl of sample buffer to the reaction and load the complete reaction onto
an 1% agarose gel. Select the lanes that contain an insert of the right size for
further analysis.
9 Overnight culture of bacteria
Inoculate 5 ml LB (with ampicilin) with the bacteria from the backup plate for
each positive clone
And grow overnight at 37°C in shaker
12
10 Isolation of plasmid DNA (TELT)
• spin down 1.5 ml cells (o/n culture) in Eppendorf, 5000rpm, 2min
• discard supernatant, add again 1.5 ml cells (o/n culture) and spin again,
5000rpm, 2min
• discard supernatant, knock onto paper towel to get rid of most of
supernatant
• add 165 µl TELT buffer, resuspend pellet completely by vortexing
• leave 5 min at RT
• 5 min 100C (get ice….)
• 5 min on ice
• Centrifuge for 10 min max speed
• remove snot pellet with toothpick, discard
• add 165 µl 2-propanol to the supernatant and mix
• Centrifuge for 15 min max speed
• discard supernatant, wash pellet in 500 µl of 70% ETOH,
• Centrifuge for 5 min max speed
• Dry DNA pellet in speedvac
• Dissolve DNA in 50 µl water
• 2 min 95-100C (kill again DNA'ses)
• Prepare spin-columns:
Load column with 850 µl Sephadex G50 matrix
Centrifuge for 2 min at 4000 rpm
Transfer column to a clean marked eppendorf tube
• Apply the sample onto the matrix in the column, Don’t touch the walls!!
• Centrifuge for 2 min at 4000 rpm
• Store DNA in fridge
11 Cycle sequencing
Use 1 µl of the plasmid DNA in the cycle sequencing reaction:
1
2
5
1
1
µl
µl
µl
µl
µl
DNA (TELT)
M13rev primer (3 µM stock)
water
Big Dye buffer
Big Dye terminator mix (PE)
cycle:
1 min
96°C
30x
10 sec 96°C
15 sec 40°C
4 min 60°C
forever
4˚C
Add 10 µl of water and bring the samples to the sequence facility on the third
floor of the Weyertal building.
12
Sequence analysis
Will be explained during course.
13
13
Preparation of DIG-labeled RNA probes for in situ hybridization
Based on protocols in TIG10 (pages 73 resp. 266)
The template you get is ready to use, this means 1 µl of 500 ng/µl of
linearized DNA or 6 µl of purified PCR product (depending on template, check
whether your template is a linearized plasmid or a PCR product). Check in the
list on the next page which enzyme (RNA polymerase) you have to use with
your template
• In vitro transcription:
1 µl
5 µl
1 µl
1 µl
1 µl
1 µl
10 µl
DNA (500 ng) (linearized plasmid)
H2O
10* reaction buffer
rNTP mix DIG labeling
RNase inhibitor (20 units)
RNA polymerase (T3/T7/Sp6)
OR
6
1
1
1
1
10
µl
µl
µl
µl
µl
µl
DNA (500 ng) (PCR product)
10* transcription buffer
rNTP mix DIG labeling
RNase inhibitor (20 units)
RNA polymerase (T3/T7/Sp6)
• Incubate 2 h at 37°C
• Add 1 µl 500 mM EDTA
• Add 90 µl H2O
• Add 1 µl tRNA (20 µg/µl)
• Add 45 µl 7.8 M NH4Ac (0.5 vol), mix, and 435 µl EtOH (3 vol)
• Precipitate 1 h at -20°C
• Centrifuge 20 min at 15,000 rpm
• Wash pellet with 500 µl 70% EtOH
• Dry in speedvac (about 5 min), or airdry.
• Dissolve in 100 µl H20
• Run 5 µl on a mini-gel (0.8% gel, 60 V, 30 min), place 25 ng template DNA
in neighboring lane. RNA band should be 5 to 10 times stronger than
template DNA.
• Store at -80°C
• Test optimal probe concentration
14
Probes for in situ Hybridizations:
Preliminary list, the final list will be provided during the course.
Cs - Cupiennius salei
Gm - Glomeris marginata
Tc - Tribolium castaneum
Dm – Drosophila melanogaster
Gene
Cs-en-1
Cs-lab
Cs-pb
Cs-Hox3
Cs-Dfd
Cs-Scr
Cs-ftz
Cs-Antp
Cs-Ubx
Cs-abd-A
Cs-Abd-B
Polymerase for anti-sense RNA
T7-RNA polymerase
T7-RNA polymerase
Gm-en
Gm-lab
Gm-pb
Gm-Hox3
Gm-Dfd
Gm-Scr
Gm-ftz
Gm-Antp
Gm-Ubx
Gm-abd-A
Gm-Abd-B
T3-RNA polymerase
T7-RNA polymerase
T7-RNA polymerase
T7-RNA
T7-RNA
T7-RNA
T7-RNA
polymerase
polymerase
polymerase
polymerase
T3-RNA polymerase
T7-RNA polymerase
T3-RNA polymerase
T3-RNA polymerase
Tc-Dfd
Tc-Scr
Tc-Antp
Tc-Ubx
Tc-en
Tc-hb
Tc-eve
Dm-en
Dm-hb
Dm-Kr
Dm-h
Dm-eve
Eppendorf tubes contain template DNA (500 ng)
For vitro transcription reactions add the remaining components to these tubes
and do the reactions in these tubes
15
14 Fixation Drosophila embryos
• Flies are in cages with an apple juice agar plate containing yeast (Flies like
this). Drosophila lays their eggs onto these plates.
• Change the plate, collect the eggs/embryos from that plate and transfer
them into an embryo basket. (egg baskets are made from polyamide
screen 0.10 mm mesh size, melted to a 1 cm section cut from a 15 ml
falcon tube: melt at 200°C on hot plate covered with aluminium foil; at this
temperature the tube should melt but not the polyamide fiber)
• Dechorionate by placing basket for 2 min into petri dish with 25% bleach
(Klorix) until dorsal appendages are dissolved (move basket with tweezers
to stir the embryos; don't overdechorionate: extended bleach treatment
results in low devitellinization efficiency!).
Be careful, bleach also bleached your cloths!!
• Rinse well with deionized H2O; transfer into scintillation vial containing 6ml
heptane, 2ml PEMS. All dechorionized embryos will be at the interphase.
Add 300µl 37% formaldehyde.
Fix for 20 min on shaking platform
• Remove aqueous (lower) phase (including non-devitellinized embryos) with
glas pipette; devitellinize by adding 8ml Methanol and shaking vigorously
for 30 sec; transfer embryos that fell to the bottom into a tube (using
pasteur pipette), discard all undevitellinized embryos at the interphase.
• Rinse twice with Methanol, store at -20oC indefinitely
15 Fixation Tribolium embryos
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Collect Embryos, put them in sieve
Rinse with de-ionized water
Wash with Klorix for 6 minutes, constantly washing the embryos with a
plastic pipette.
Change the Klorix when it becomes cloudy
Rinse with de-ionized water
Dry embryos with a tissue
Prepare scintillation vials with 6 ml Heptane, 3 ml PEMS (solutions list)
Transfer Embryos to scintillation vial with brush
add 350l Formaldehyde (37%) to each vial
Fix for 40 minutes on shaker
Remove lower phase (PEMS & Formaldehyde) with a glas pipette
Add 8 ml Methanol and shake immediately for at least 1 minute, vortex
The devitellinized embryos are now at the bottom of the vial. Collect them
in a falcon tube with Methanol.
Aspirate with syringe several times to devitelinize the remaining embryos.
Collect them again
Wash twice with Methanol
Store at –20˚C
16
16
Whole mount In-situ hybridization -- Tribolium & Drosophila
All reactions are done in Eppendorf vials. Take 20-50l settled embryos per
vial
R= rinse, swirl with 1 ml of fresh solution, let embryos settle
RR= rinse 2x
W5= wash on the wheel for 5 (10, 15) minutes
•
•
•
•
•
•
•
•
•
•
•
•
R with 50% Methanol, 50% PBT
R with PBT
Post-fix in 1ml PBT +140l formaldehyde (37%) for 15 min on wheel
RRRRR with PBT
Incubate 5 min in 1 ml PBT with Proteinase K (For Tribolium: use 2.5l of
a fresh 1:10 dilution; for Drosophila use 5l of fresh 1:10 dilution) DON'T
OVERDIGEST: roll vials for 4 min, then let embryos settle down for 1 min,
Replace IMMEDIATELY with PBT
RR with PBT
Post-fix again in 1 ml PBT + 140l Formaldehyde (37%) for 15 min on
wheel
RRRR with PBT
Rinse with 250l PBT + 250l Hyb-B
Replace with 250l Hyb-B
Replace with 250l Hyb-A, preincubate in waterbath at 65°C for 1 hour
Aspirate as much Hyb-A as possible, add probe (1-5l) diluted in 30l Hyb-A
---------------Hybridize overnight in 65°C waterbath----------------------•
•
•
•
•
•
•
•
add 500l Hyb-B (prewarmed to room temperature), keep at 65°C until
embryos settle down.
Replace with 500l Hyb-B, incubate at 65°C for 15 min
proceed at room temperature: add 500l PBT
RR W15 W20 with PBT + 10mg/ml BSA + 2% Sheep Serum
Rotate for 1 hour with 1 ml anti-Dig antibody on wheel (dilution 1: 2000 in
PBT)
RRR W20 W30 W30 WITH PBT
RR with Staining Buffer (freshly prepared)
Stain in 1ml Staining Buffer + 20 l NBT - X-phosphate mixture, do not
rock, keep embryos in the dark. Staining may take 30 min to several
hours. Stop reaction by washing with PBT several times, store embryos at
+4°C in PBT
17
17
Whole Mount in situ Hybridization for the spider
Cupiennius salei.
Damen and Tautz (1998) Dev. Genes Evol. 208:586-590, Adapted from the
Drosophila protocol (Tautz and Pfeifle 1989 and Klingler and Gergen 1993)
Please Note: The spider embryos you get are already fixed and are in MeOH
Fixation
• Dechorionate embryos in 50% Klorix (typically 1-2 minutes)
• Wash embryos several times in H2O
• Fix embryos with 5% formaldehyde in PEMS and heptane (1:1) for several
hours to overnight on the wheel at RT.
Alternatively, use 4% paraformaldehyde in PEMS and heptane (1:1)
[prepare fixative fresh, adjust pH to 7.0].
• For young stages: Fix embryos in formaldehyde saturated heptane (add
200 µl of formaldehyde to 10 ml of heptane, shake vigorously for 10 min.,
add this mixture to the dechorionated embryos) Do not put the embryos
onto the wheel. Fix for several hours to overnight at RT
• Remove fixative as complete as possible and add carefully 100% MeOH
• Wash the embryos once with 100% MeOH (carefully!!)
• Let stand for 30 min to overnight at RT and store at –20˚C
• Remove vitellin membranes using watchmaker forceps (Dumont 5)
• Transfer the embryos to 2 ml Eppendorf tubes in 100% MeOH
• Replace with fresh MeOH
• Store the embryos at -20°C for at least 30 minutes.
Embryos can be stored in this way for months.
• Return embryos to RT
• Immerse 5 min in 50% MeOH in PBS-T
• Immerse 5 min in 30% MeOH in PBS-T
• Rinse three times in PBS-T for 5 min each
• Fix for 20 min in 5% formaldehyde in PBS-T at RT (1 ml PBS-T + 170 µl
formaldehyde).
• Rinse three times in PBS-T for 5 min each
Proteinase digestion and post-fixation
• Digest with proteinase K
use 3.5 µl of a 1:10 dilution per ml of PBS-T, at RT for 4 minutes (depends
on the stage, younger stages are more sensitive; depends also on the batch
of enzyme)
• Rinse briefly in PBS-T
• Wash twice in PBS-T for 5 minutes
• Fix for 20 min in 5% formaldehyde in PBS-T at RT
• Wash three times in PBS-T for 5 min each
• Wash embryos once in PBS-T - HYB-B (1:1) for 5 min (1 ml)
Prehybridization
• Incubate embryos in 500 µl HYB-B for 5 minutes at 65°C
• Replace HYB-B with 500µl of HYB-A
• Prehybridize at 65°C for 1 h in HYB-A
Hybridization
• Remove as much HYB-A solution as possible without letting the embryos
touch air
• Add 50 µl of fresh HYB-A containing 5 µl of RNA probe, so that all embryos
are covered by the solution.
• Incubate overnight at 65°C
18
Probe removal
• Remove as much as possible of the hybridization solution
• Add 500 µl HYB-B and incubate at 65°C for 10 min
• Replace with 500 µl fresh HYB-B and incubate at 65°C for 30 min
• Replace with 500 µl 50% HYB-B + 50% PBS-T and incubate at RT for 10 min
• Wash embryos twice with PBS-T (5 min each)
• Wash embryos twice with PBS-T (15 min each)
• Additional PBS-T wash steps are allowed. (30 min.)
• Incubate in PBS-T supplemented with 10 mg/ml BSA and 2% sheep serum
for 2 x 30 min
Detection
• Add the anti-DIG antibody (Fab-AP as supplied by Roche) to a final dilution
of 1:2000 in PBS-T supplemented with 10 mg/ml BSA and 2% sheep serum
(12.00)
• Incubate for 3 - 4 h at RT
• Wash twice for 10 min each with PBS-T
• Wash twice for 30 min each with PBS-T
• Additional PBS-T washes: No problem!!
• Wash o/n with PBS-T at 4°C
• Wash 3 times for 5 min each in staining buffer
• Incubate in staining buffer with 20 µl NBT/X-Phosphate mixture per ml
• Stain for as long as you like to stain (in dark)
• Stop staining reaction by several washes in PBS-T
• Add one drop of formaldehyde
• Store embryos at 4°C in PBS-T
• Wash embryos two times with PBS-T (to remove formaldehyde) before
making drawings of the embryos
19
Solutions for in situ hybridisations
PEMS
0.1M Pipes
2mM MgSO4
1mMEDTA
pH 6.9
For 800 ml:
24.16 g Pipes, 1600l 1M MgSO4, 1600l 0,5 M EDTA
PBS (10x)
80g NaCl
2 g KCl
2 g KH2PO4
11.5g Na2HPO4
Fill to 1000 ml, pH. 7.4
autoclave
PB-T Tribolium/Drosophila (PBS + 0.02% Tween 20)
80 ml 10x PBS
1.6 ml 10%Tween 20
Fill to 800 ml, pH 7.4
PBS-T spider (PBS + 0.02% Tween 20)
40 ml 10x PBS
0.8 ml 10%Tween 20
Fill to 400 ml, pH 7.4
20 x SSC
 70.12 g NaCl
 35.28 g Na Citrate
Fill to 400 ml, pH. 7.0
add 200 l DEPC, autoclave
Hyb-A (store at -20 for up to 3 months)
 25 ml formamide (aliquotes in freezer)
 12.5 ml 20x SSC (pH 7.0)
 1 ml of 10mg/ml salmon testis DNA (aliquotes at –20˚C)
 250 l 20mg/ml tRNA (aliquotes at –20˚C)
 25 l 100mg/ml heparin stock (aliquotes at –20˚C)
 0.1 ml of 10% stock Tween-20
Fill to 50 ml with water
Hyb-B
 25 ml formamide (aliquotes in freezer)
 12.5 ml 20x SSC (pH 7.0)
 0.1 ml of 10% stock Tween-20
Fill to 50 ml with water
Staining Buffer (prepare fresh)
 5 ml 1 M Tris pH 9.5
 2.5 ml 1 M MgCl2
 1 ml 5 M NaCl
 0.1 ml 10% Tween 20
Fill to 50 ml with water
20
Anhang 1
The Genetic code
Anhang 2: IUB code for mixed base sites:
N
V
B
H
D
K
S
W
M
Y
R
A
A
C
A
A
G
C
A
A
C
A
C
C
G
C
G
T
G
T
C
T
G
G T
G
T
T
T
21
Anhang 3: Amino acids
22
Anhang 4 Gene names
Maternal effect genes
bcd
bicoid
Gap genes
hb
hunchback
Kr
Krüppel
gt
giant
kni
knirps
slp
sloppy paired
otd
orthodenticle
cad
caudal
Pair rule genes
h
hairy
eve
even-skipped
run
runt
ftz
fushi tarazu
prd
paired
opa
odd paired
odd
odd skipped
slp
sloppy paired
Segment-polarity genes
en
engrailed
wg
wingless
gsb
gooseberry
ci
cubitus interruptus
hh
hedgehog
Hox genes
lab
labial
pb
proboscipedia
Hox3
Hox3
Dfd
Deformed
Scr
Sex-comb-reduced
Antp
Antennapedia
Ubx
Ultrabitorax
abd-A abdominal-A
Abd-B Abdominal-B
23
Anhang 5:
24
Anhang 6: DNA Marker (1 kb plus Invitrogen)
25