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[Introduction]
[Method I: choosing ODN] [Method
II: ODN modification] [Method III: host transfer
technique] [Method IV: fertilization and development]
[Appendix + solutions]
| Oocyte
transfer: Appendix and Solutions |
Protocol
submitted by: |
A)
Solutions for maintenance and manipulation of Xenopus oocytes and embryos.
OCM
(Oocyte Culture Media)
480 ml Liebovitz L-15 medium (Sigma L-5520)
320 ml sterile deionized water
0.32 g Bovine serum albumin (BSA) (Sigma A-9418)
4 ml Glutamine from 200 mM stock (Sigma G-6392) (make fresh weekly)
4 ml Penicillin-Streptomycin solution (Sigma P-0781)
adjust pH to 7.6-7.8 with 5M NaOH
Make fresh daily |
10X
MMR (MarcÕs Modified RingerÕs Solution)*
1M NaCl
20mM KCl
20mM CaCl2
10mM MgCl2
150 mM Hepes*
pH 7.6 store at 4 degrees Celsius
*This recipe is actually a modification of MMR.
We use 3x the normal Hepes concentration. |
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Anesthetic
2 gm/liter 3-Aminobenzoic Acid Ethyl Ester (Sigma A-5040) in Amquel treated
water |
Progesterone
solution for maturing oocytes
1 mM Progesterone (Sigma P-0130) in 100% Ethanol |
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B) Vital dyes.
When
making up vital dyes be aware that different batches of dyes stain oocytes
differently. Therefore it is necessary to visually inspect oocytes and determine
how much vital dye is required to stain the oocytes each of the 5 colors in
the 15 min. incubation used to dye the oocytes. Although we have attempted
to make more than 5 colors using combinations of vital dyes, only 5 are easily
distinguishable:
blue... 0.1% Nile Blue A (Sigma N-0766)
red...0.25% Neutral Red (Sigma N-6634)
brown...2% Bismarck Brown (Sigma B-2759)
green (100 µl blue + 100 µl brown)
mauve (100 µl blue + 100 µl red).
Red,
blue, and brown dyes are dissolved in distilled sterile water and spun down
after shaking for 0.5 hours. The supernatant is aliquotted as 1 ml aliquots
in 1.5 ml microfuge tubes. The pellet is discarded. Dyes are added directly
to the OCM containing the oocytes. We use 100µl of red, blue or brown dyes
in 8ml of OCM. Green is obtained by adding 100µl of blue and brown, and purple
(mauve) by mixing 100µl of blue and red. Oocytes are colored for approximately
15mins on a rocking platform, and then washed in a large dish of OCM.
[Introduction]
[Method
I: choosing ODN] [Method II: ODN modification]
[Method III: host transfer technique] [Method
IV: fertilization and development]
[Appendix + solutions]
| 
[2.3.1]
