Truelab

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1. Combine the following in a 0.2ml PCR tube:
250-500ng DNA/sample,
2.2pmol primer/sample,
add PCR water to total volume of 8ul.

2. Add 4ul per sample of Master Mix (see recipe below).
Big Dye v3.1: 2 ul
Big Dye dilution buffer 2 ul

Step 1 Incubate at 96.0C for 20 seconds
Step 2 Incubate at 50.0C for 20 seconds
Step 3 Incubate at 60.0C for 4 minutes
Step 4 Repeat to Step 1 for 39 times
Step 5 Incubate at 12.0C forever
Step 6 End

4. Clean up the reaction with various methods.
Quantity of template DNA varies depending on the size of fragment.

Template size Quantity 100-200 bp 15-30 ng 500 bp 30-65 ng 1000 bp 65-130 ng 2000 bp 125-250 ng 3000 bp 250-500 ng

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(1) Homogenize 50 adults in a 1.5 ml eppendorf tube with 500 ul DEB buffer and 225 ul phenol [wear gloves and goggles] using blue pestil and battery powered grinder until well homogenized (>= 1 min). Transfer to new eppendorf tube. Rinse old tube with 225 ul phenol and add to new tube. Vortex gently.

(3) Remove aqueous layer (top) avoiding interface to new eppendorf tube. Add 500 ul each of chloroform (24:1 with isoamyl alcohol) and phenol. Vortex gently but well.

(5) Remove aqueous layer (top) avoiding interface to new tube. Add 500 ul chloroform. Vortex gently but well.

(7) Remove aqueous layer (top) avoiding interface to new eppendorf tube. Measure volume using pipettor. Add 1/10 of this volume 3M Sodium Acetate and then 2.2 times the total new volume 100% EtOH. Chill at –20oC for 15 minutes or –70oC for 3 minutes.

(9) Decant EtOH and wash pellet (or smear) in 250 ul of cold 70% EtOH. Decant EtOH and invert tube on kimwipe. Air dry.

(10) Resuspend pellet in 100 ul TE pH 7.4. If pellet has trouble resuspending, place at 65oC (or 50oC) for 20 minutes.

(13) Remove aqueous (top) layer to new tube. Add 50 ul each of phenol and chloroform. Vortex gently. Centrifuge 5-10 minutes at 15K RPM.

(14) Remove aqueous (top) layer to new tube. Add 100 ul chloroform. Vortex gently. Centrifuge 2-5 minutes at 15K RPM.

(15) Remove aqueous (top) layer to new tube. Measure volume with pipettor. Add 1/10 of this volume 3M Sodium Acetate and 2.2 times the new total volume 100% EtOH. Place at –20oC for 15 minutes or –70oC for 3 minutes.

(17) Decant EtOH and wash pellet in 250 ul of cold 70% EtOH. Decant EtOH (pipette off all alcohol) and invert tube on kimwipe. Air dry.

(18) Resuspend prep in 50 ul TE pH7.4. If pellet has trouble resuspending, place at 65oC (or 50oC) for 20 minutes.

DEB Buffer (Make fresh before preps. Do not store more than 2 days. Do not refrigerate.)

	10 ml	25 ml	50 ml
SDS	0.05 g	0.125 g	0.25 g
5M NaCl	400 ul	1 ml	2 ml
0.5 M EDTA	500 ul	1.25 ml	2.5 ml
1M Tris 8.0	100 ul	250 ul	500 ul
sterile dH2O	9 ml	22.5 ml	45 ml
Final Volume	10 ml	25 ml	50 ml

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QUARANTINE STOCKS
The following is a general outline of steps which should be followed when new stocks are brought into the lab. It is important that stocks go through the entire quarantine; procedure to prevent a mite infestation. To be safe, one should order stocks from Bloomington or Bowling Green Stock Center's whenever possible. Stocks should never be ordered from a lab known to have mites. If you don't know if a particular lab has mites, ask someone before you order it.

When handling quarantine stocks, it is a good idea to treat them as if there were mites present. Therefore, all stocks should be kept in vials with tight cotton plugs, not bottles. New vials should be set up shortly after the adults eclose, and most importantly, quarantine stocks must be kept isolated from all other stocks.

1. New stocks must be brought directly to an isolated spot. Do not bring them near any lab stock.
2. At a quarantine microscope, transfer the adults to a fresh vial (P0) and keep the original vial for a few days.
3. When it is obvious that the P0 vial is going fine, discard the adults and the original vial.
4. Shortly after the adults eclose from the P0 vial, transfer them to a fresh vial (F1).
5. Keep the stock isolated for two more generations (F1 and F2).
6. After you have set up the F2 vial, check the P0 vial for mites.
7. When the adults eclose from the F2 vial, put them in a fresh vial and check the P0 and F1 vial for mites.
8. If no mites are found, the stock may be taken out of quarantine.
9. If mites are found at any time or suspected of being in a stock, transfer the adults from the suspect vial into a vial containing a Tedion strip (strips are made by soaking filter paper in a 0.5% (w/v) solution of Tedion in acetone and then allowed to air dry).
10. Autoclave the old vial.
11. Every two days, transfer the adults to a new vial containing Tedion and autoclave the old vial. Continue this for one week.
12. Place adults in a new vial containing Tedion and follow steps 3-7 as outlined above. The only exception is that you allow two additional generations (F3 and F4) in isolation.

The Curator of Drosophila Stocks
Dept. of Biology
Jordan Hall A503
Indiana University
Bloomington, IN 47405
(812) 855-5783
(812) 855-2577 (FAX)
Kathy Matthew, Curator (812) 855-5782

Mid-America Drosophila Stock Center
Dept. of Biological Sciences
Bowling Green State University
Bowling Green, OH 43403
(419) 372-2631

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(2) Cut pupae longitudinally (either into left-right or dorsal-ventral halves as desired) and use forceps to transfer hemipupae into a 1.5 ml eppendorf tube containing 1 ml of PBS+.3% Triton X-100 (PBST)[150 ul Triton X-100 in 50 ml PBS]

(4) Add formaldehyde to a final concentration of 4%. [add 108 ul of 37% formaldehyde stock to each tube]

(5) Place on a gentle shaker/rocker at room temp for 40 minutes. Add 50ul 0.5% methylene blue to stain the cells and rock for 5 more minutes.

(6) Dissect away remaining fat cells, muscles, pupal cuticle/case in PBS on a microscope slide.

(7) Block in PBST+3% BSA (PBSTB) at 4oC for 1 hr to overnight. [add .45g of BSA to 15 ml of PBST]

(8) Incubate with primary antibody (1:200 rabbit anti-Yellow, 1:1000 rat anti Bab2 in PBSTB) at 4oC overnight with rotation.

(9) Save primary antibodies and wash four times for 15 minutes with PBST at 4oC with rotation

(10) Incubate with secondary antibodies (1:200 anti rat Cy3, 1:200 anti-rabbit FITC in PBSTB) for 4 hours to overnight at 4oC with rotation.

(11) Wash four times for 15 minutes with PBST at 4oC with rotation. Include TOPRO 2 nuclear staining reagent at a concentration of 1:2000 in the first three washes.

(12) Mount pupal abdomens in vectashield and store at 4oC in dark until confocal microscopy.

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Protocol adapted from HYPERLINK "https://dna.biotech.wisc.edu/documents/Non-bead_cleanups.htm" https://dna.biotech.wisc.edu/documents/Non-bead_cleanups.htm

1. Sephadex G-50 DNA grade (fine; available through many distributors including Pharmacia and Sigma. G-50 Med or Fine also works)
2. Columns (96-well or used AutoSeq G-50 columns from Amersham).
3. Autoclave
4. Centrifuge

2. Weigh out the sephadex and put it in an autoclave safe glass bottle.
(use 2 g. Sephadex G-50 with 80 ml. of water)

3. Autoclave the mixture for 15 minutes and store in a refrigerator.
Note: Autoclaving will speed up the hydration of the sephadex and kill any fungus that may show up if the slurry is not used immediately.

4. Decant excess water after autoclaving so that there is approx. 1/3 water with 2/3 sephadex.

5. Transfer 750 ul of well shaken slurry to the top of each of the columns.
Note: Make sure the slurry is thoroughly mixed and uniform before adding it to a column. If this is not done, the sephadex “pillars” may not be uniform and the results from the sample clean-up may not be good.

6. Spin down the G-50 column at 2000xg (5000 rpm in Eppendorf microcentrifuge) for 1 minute to remove the water (if using a standard microfuge with variable speeds a setting of 5 out of 14 works well). Spin the 96-well plates at 750xg for approximately 2 minutes.
Note: The columns should be spun in a tube that it will collect the water from the hydrated sephadex. A waste tray should also be used for the 96-well tray.

7. Transfer the columns to either the sample collection tray (96-well) or a 1.5-ml labeled microfuge collection tubes.

11. The remaining resin can be tapped out and the column re-used indefinitely after washing with tap water and rinsing with dH2O. For the 96 well columns, it may be necessary to spray out some of the chambers to remove all the used matrix.

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(1) Freeze flies at –20oC, one fly per 1 ml eppendorf tube (alternatively collect flies fresh and place on ice).

(2) Make a cocktail containing 250 ul of homogenization buffer plus 2.5 ul Proteinase K (20 mg/ml stock in –20oC freezer) for each fly sample. E.g. for 10 flies, mix up 2500 ul homogenization buffer (2.5 ml) plus 25 ul Proteinase K stock in a Falcon tube.

(3) Add 250 ul of homogenization buffer/Proteinase K cocktail to each tube containing a fly.

(4) Homogenize each fly thoroughly using clean blue pestle. You should grind for 30 sec to 1 min or until the solid material appears well homogenized. BE SURE TO CHANGE PESTILS BETWEEN FLIES.

(9) Centrifuge 14K rpm (max) 10 min. Be sure to line the tabs of the tubes up around the outside of the rotor so that if your pellet is invisible you will know where it is.

(10) The pellet may or may not be visible. Pipette off the Ethanol being careful not to disturb the area of the pellet.

(11) Wash pellet by pipetting in 500 ul cold (-20oC) 70% Ethanol. Use the P200 pipette to remove as much liquid as possible.

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