Arrival of Xenopus laevis sperm samples
The frozen sperm samples should arrive at the lab in a sealed polystyrene box covered in dry ice. Samples must remain frozen and should be placed immediately toward the back of a -80oC freezer until use.
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Arrival of Xenopus laevis sperm samples
The frozen sperm samples should arrive at the lab in a sealed polystyrene box covered in dry ice. Samples must remain frozen and should be placed immediately toward the back of a -80oC freezer until use.
Notes:
1Before starting ensure that a water bath is preheated to 37oC, 0.1xMBS is at room temperature and that you have large orifice or pre-cut 20-200µL tips available. We usually assess the quality of a small clutch of unfertilised eggs prior to defrosting the sperm. When recovering frozen sperm stored in distant -80oC freezers it is recommended that it is first transported on dry ice to the fertilisation bench before continuing with the protocol.
2frozen sperm sediments, pipette gently up and down to maintain in suspension.
3 If eggs are clustered and layered, gently shake the Petri dish or separate egg layers using pipette tips prior to incubating. An additional wash step (125 µL 0.1 x MBS) of the vial containing sperm is recommended for maximum efficiency.
Equipment
Note: a hot block is not a suitable alternative
Materials
Note: it is recommended that standard tips, if used, should be cut using a razor blade to enlarge the orifice preventing sperm damage
Reagents
Modified Barth’s Saline (Gurdon, 1977)
Barth-HEPES Saline (10X): Weigh 51.3 g NaCl (e.g. Sigma, S7653), 0.748 g KCl (e.g. Sigma, P9541), 2.02 g NaHCO3 (e.g. Sigma, S6297), 23.832 g HEPES (e.g. Sigma, H4034) and dissolve in 800 mL de-ionised water. Add 5.6 mL 5 M NaOH (e.g. Sigma, S5881), 2.02 g MgSO4 (7H20 – e.g. Sigma, M2773), 0.920 g Ca(NO3)2 (4H20 – e.g. Sigma, C1396) and 0.603 g CaCl2 (2H20 – e.g. Sigma, C5080). Make up to a final volume of 1 L using de-ionised water and filter sterilise (or autoclave), the pH of the final solution should be around pH 7.6. Store MBS at 4oC and check for salt precipitation prior to use.
Generate 1 x MBS and 0.1 x MBS using de-ionised water. To 1 L of 1 x MBS or 0.1 x MBS, 1 mL penicillin-streptomycin solution (P/S – e.g. Sigma, p0781) is added to generate a final working concentration of 5 U/mL penicillin and 5 µg/mL streptomycin.
Cysteine
Mix 2 g Cysteine per 100 mL de-ionised water, stir until dissolved, adjust to pH 7.8-8.0 by adding NaOH (e.g. Sigma, S8045) and store in an air-tight container until use.