Protocols

 
Brdu Staining

Extract

Cpd

Fly Food

Gel Electrophoresis

Ligation
 

    

BrdU   Staining

1) Dissect flies and separate ovaries into embryo dish with 3 mL Grace's medium.
2) Remove ovaries from Grace medium into 1 mL BrdU Solution (1 mg/mL BrdU in Grace medium). Incubate for 1 hour at room temperature.
3) Remove BrdU solution and discard in BrdU waste. Fix ovaries in 500 µL FIX solution (EM grade formaldehyde, Buffer B, and H2O at 2:1:3) for 10 minutes.
4) Remove FIX and discard in formaldehyde waste. Wash with PBT twice, 15 minutes each.
5) Remove PBT and discard in sink. Wash ovaries twice with 1 mL DnaseI reaction buffer, 15 minutes each.
6) Remove DnaseI reaction buffer and discard in sink. Add 6 µL DnaseI in 500 µL DnaseI reaction buffer.  Incubate for 30 minutes at 37° C. Remove DnaseI and buffer and discard in sink.
7) Wash 3 times with PBT, 1 minute each.
8) Wash once for 30 minutes with 1 mL PBT.
9) Remove PBT and discard in sink. Block with 1 mL PBTG for at least one hour.
10) Remove PBTG and discard in sink. Stain with 150 µL anti-BrdU antibody overnight in 4° C room.
11) Remove anti-BrdU. Wash with 1 mL PBT three times, 15 minutes each.
12) Remove PBT and discard in sink. Add 150µL secondary antibody and nutate overnight at 4° C.
13) Remove secondary antibody. Wash twice with PBT, 15 minutes each.
14) Remove PBT and discard in sink. Add 150 µL DAPI solution and nutate for 10 minutes.
15) Remove DAPI. Wash once with 1 mL PBT for 10 miniutes, then once with 1 mL 1XPBS for 10 minutes.
16) Remove PBS and discard in sink. Add mounting solution and mount ovaries on slide.

PBT (100 mL):

10x PBS 10 mL
20% Triton X-100 2 mL
dH2O 88 mL

BrdU Buffer B:

KH2PO4 (pH 6.8) 100 mM
KCl 450 mM
NaCl 150 mM
MgCl2 20 mM

DnaseI reaction buffer (50 mL):

1M Tris (pH 7.5) 3.3 mL
2M MgCl2 0.125 mL
2-ME (98%) 4.0 mL
dH2O 46.575 mL

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Genomic DNA Prep

1) Collect 30 anesthetized flies or larvae in a 1.5 mL Eppendorf tube and place on ice.

2) Homogenize flies in 400 µL of Fly Squishing Buffer A, continue to grind for 1-2 minutes

3) Incubate at 65° C for 30 minutes

4) Add 800 µL Fly Squishing Buffer B to each smaple, mix well by inverting the tube multiple times. Incubate on ice for at least 10 minutes.

5) Centrifuge for 15 minutes at 12,000 rpm at room temperature

6) Transfer 1 mL of the supernatant to a new 1.5 mL Eppendorf tube. Be careful to only remove the supernatant, and not any residual precipitate that may not have pelleted

7) Add 600 µL of ice-cold 100% ethanol to each sample and mix well by inverting the tube several times

8) Centrifuge for 15 minutes at 12,000 rpm at room temperature

9) Discard the supernatant - carefully add the remaining portion of the previous supernatant along with another 600 µL ice-cold 100% ethanol

10) Centrifuge again for 10 minutes at 12,000 rpm at room temperature

11) Discard the supernatant - Wash the pellet with 70% ethanol, dry for two minutes using a vacuum centrifuge, and re-suspend DNA in 150 µL 1X TE pH 7.0

12) Store at -20° C until use

This protocol is taken from "Drosophila Protocols" by Sullivan, Ashburner, and Hawley.

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Critical Point Dry Prep

- anesthetize flies with CO2 and gently remove head with double-edged razor blade
- use a paint brush to transfer the individual heads to the 9-sectioned CPD holder (at least 5 heads per section) and seal the top
- pour 25% ethanol into a baby food jar (about 3/4 full) and quickly place the CPD holder into the ethanol and seal

* It is imperative from this point that the samples are not allowed to contact air long enough to dry. This will ruin the surface features.

- allow the sample to sit in the 25% ethanol at room temperature overnight
- using a second baby food jar prepare a similar amount of 50% ethanol and quickly transfer the CPD holder from one jar to the other
- allow the sample to sit in 50% for 30 minutes
- continue this procedure with transfer to 75% ethanol once, and 100% ethanol twice (the 100% ethanol must be from a previously unopened container)
- once the samples are in 100% ethanol, they are stable for up to one month before critical point drying, however it is recommended to dry ASAP


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Food Recipe

* Prepare trays of empty food vials/bottles and setup dispenser before beginning *

* A single batch of food will make approximately 2 1/2 trays of vials *

- Measure out 2.7 L dH2O, begin heating on burner

- Measure 180 g malt extract and 300 mL light corn syrup in separate containers

- Measure 67.5 g yeast and 39 g soy flour, combine together and add 300 mL d H2O, mix until smooth

- Measure 285 g corn meal and 22.5 g agar, combine together and add 500 mL dH2O, mix until smooth adding more water   as necessary

- Once water is boiling, pour in cornmeal/agar mixture, making sure to stir continuously

- Allow the mixture to heat to a boil again, during this time, break any lumps that may have formed

- Once mixture beings to bubble on edges, stir in malt (don't worry about the lumps of malt, they will cook out)

- Add yeast/soy flour mix and then corn syrup, stir well

- Allow mixture to heat, once bubbling is seen on the edges, cook an additional 10 minutes, stirring frequently

- After 10 minutes, add 19 mL propionic acid and a small amount of appropriate antibiotic, stir well

- Remove from heat and dispense immediately

- Allow to cool at room temperature before capping, leave capped vials at room temperature overnight

- Place vials in 4 degree cold room for storage

 

* This recipe is the same one used by the Bloomington Stock Center

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Gel Electrophoresis

Making the gel:

- a 1% gel is 0.5 g agarose in 50 mL dH2O
- the gel percentrage is based on the size of fragments being seperated; the smaller the fragments, the higher the gel   percentage
- the two ingredients should be combined in a small capped glass container and microwaved until the first boil (don't forget   to loosen the cap while heating)
- after mixing, recap (loosely) and bring to second boil
- remove from the microwave, and wearing gloves, add an appropriate amount of ethidium bromide (EtBr) to the gel
* EtBr stock should be added as 100 µL to 100 mL gel *
- tape the sides of the gel plate with Scotch tape and trim the excess with a razor blade
- once the gel is cool to the touch, pour into gel plate, make sure that gel does not melt the tape, add a gel comb
- once the gel has cooled and set, remove the tape and pour appropriate concentration of TBE of gel until it is just covered
- carefully remove the comb, being sure not to break the wells

Loading the gel:

- using Parafilm, make indentations for each sample in a tube block
- add each component to the indentations, then use a tip to take up and dispense several times to mix the sample
- load into the gel, remembering to leave the first well open for a DNA ladder
- attach a cover to the gel apparatus, RUN TO RED!!! remember that DNA is negatively charged and will run to the positive   pole
- run the gel at approximately 100 V for shortly over 1 hour
- bromophenol blue (BPB) in the ladder and gel loading buffer will run alongside the 500 bp DNA band as a reference

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Digestion and Ligation

Digestion:

- 10 µL genomic DNA (from approximately 2 flies)
- 2.5 µL 10X buffer
- 2.0 µL 100 µg/mL RNase
- 8.0 µL dH2O
- 1.0 µL enzyme

1) incubate 2.5 hours at 37° C
2) heat for 20 minutes to 65° C

Ligation:

- 10.0 µL digested genomic DNA
- 80.0 µL 5x ligation buffer
- 310 µL dH2O
- 2.0 µL ligase

1) incubate overnight at 4° C
2) add 2.5 volumes 100% ethanol and 0.1 volume sodium acetate and precipitate on ice for 10 minutes
3) spin, aspirate, pulse, aspirate
4) speed vac briefly to dry pellet
5) resuspend in 150 µL 1x TE

 

* This protocol taken from Berkeley Drosophila Genome Project resources *

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