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INFORMATION FOR

Protocols

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0.1 M CaCl2
Dissolve CaCl2 11.1 g/liter.
Autoclave and store at -20ºC or 4ºC.
Typically prepare 200 ml.
1/9X MR + 1% Gentamicin
For 1L:
111 ml 1X MR + 889 ml DI water
Add 1000µl 1000X gentamicin stock
Use within 1 week
1X Modified Ringer's Solution (1X MR)
0.1M NaCl
1.8 mM KCl
2.0 mM CaCl2
1.0 mM MgCl2
5.0mM Hepes pH 7.6
Dissolve in ddH20 to desired volume, adjust pH to 7.8 - 8.0 with NaOH.
Use within 1 month.
1X MBS (Modified Barth's Saline), pH 7.8
Need 0.1 M CaCl2 and 10X MBS salts, pH 7.8
Mix 100 ml of 10X MBS salts with 7 ml of 0.1 M CaCl2.
Adjust volume to 1 liter with distilled water, adjust pH to 7.8.
Store at RT.
1X MBS + 0.1% BSA
For 200 ml: Dissolve 0.4 g of BSA in 180 ml of 1X MBS solution.
Adjust volume with 1X MBS. Check pH 7.8. Filter sterilize.
Aliquot to 50 ml conicals. Keep refrigerated.
"In Use" aliquot is kept at RT.
Avoid contamination of solution. Discard if solution becomes cloudy.
2.5% Ficoll in 1/3X MR (X.laevis)
Dissolve 2.5 g Ficoll/100ml 1/3X MR and autoclave (Takes a long time to dissolve.)
Store at RT.
Avoid contamination, discard if solution becomes cloudy.
2.5% Ficoll, in water
2.5 g ficoll/100ml DI water (usually make 500 ml)
Autoclave and store at RT.
Use in place of water in dish to stabilize embryos.
3% Ficoll in 1/9X MR (X.tropicalis)
Dissolve 3.0 g Ficoll/100ml 1/3X MR and autoclave (Takes a long time to dissolve.)
Store at RT.
Avoid contamination, discard if solution becomes cloudy.
100X Benzocaine Stock
Sigma Cat #E1501-500g (500 g tub)
100X stock (5%) = 5 g/100 ml 100% ethanol, Store at 4 degrees
Dilute to 1X working solution with DI H20. pH working solution to 6.8 - 7.0 with NaHCO3. Store at -20C
Note: we use Tricaine 2g/L, ph 7.0 as an anesthetic post stage 23.
10X PCR Reaction Buffer

Preparing 10X PCR Reaction Buffer + MgCl2

100 mM Tris-HCl pH 8.3

15 mM MgCl2

500 mM KCl, pH 8.3 (20°C)

To make 1 Liter:

  • 12.1 g Tris
  • 37.27 g KCL
  • 15 ml MgCl2 1M solution

Add reagents to bottle, add 800 ml DI water. Titrate pH to 8.3. Add DI water up to 1000 ml volume.

Aliquot and store at -20C.

10X MBS salts
For 1 L:
NaCl 51.43 g (880 mM)
KCl 0.7455 g (10mM)
MgSO4 1.204 g (10mM)
50 ml 1M Hepes, pH 7.8 (50 mM)
NaHCO3 2.1g (25 mM)
Dissolve in dH2O to 1L, adjust final pH to 7.8 with NaOH, autoclave. Store at RT.
Agarose plates, 1%(for use with trops or laevis)
1g agarose powder + 100 ml 1/9X MR (no gentamicin) in 500 ml bottle.
Note: Make no more than 300 ml (to prevent boil over).
Microwave on high for about 2 min/100 ml (w/ loose lid). Swirl and check for solids every 30 seconds (use hot mitts).
When agarose is completely melted, fill petri dish 1/4 full.
Put in 4C for 5 min, or cure at RT for 30 min.
Note: Insert any well molds (to hold embryos in desired position) into surface of agar when it is slightly cured. Freeform wells can be made after agar cures fully using heated forcep tips, bacterial loup etc. to melt the agarose.
Flood agar with 1/9X MR for trop embryos or 1/3X MR for laevis embryos. Keep covered with MR and a lid to prevent drying out.
Alkaline Phosphatase (AP) Buffer, with Levamisol and Tween
For 1 L:
100 ml 1 M Tris pH 9.5
50 ml 1 M MgCl2
25 ml 4 M NaCl
1 ml Tween-20
2 ml 1 M Levamisol
Adjust volume, store in 50 ml aliquots at -20C.
Amphibian Ringers, 1 L
NaCl 6.6g
KCL 0.15g
CaCL2 0.15 g
NaHCO3 0.2 g
DI water to 1 L
The solution may be stored for up to 7d, or longer if it is autoclaved and stored in sterile containers.
Antibody Staining - Acetylated Tubulin for epidermal cilia
Antibody Staining-General Protocol
Antisense RNA Probe(dig labeled) - Long
Antisense RNA Probe(dig labeled) - Short