HAT Selection Medium
Hypoxanthine, aminopterin, and thymidine selection (HAT) medium is commonly prepared from two stock solutions, 100× HT and 100× A.
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1. To prepare 100 mL of 100× HT, dissolve 136 mg of hypoxanthine and 38 mg of thymidine in 100 mL of H2O suitable for tissue culture. Heat gently (70°C) if they do not dissolve completely.
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The 100× stock is 10 mm hypoxanthine and 1.6 mm thymidine.
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2. To prepare 100 mL of 100× A, add 1.76 mg of aminopterin to 100 mL of H2O suitable for tissue culture. Add 0.5 mL of 1 n NaOH to dissolve. Titrate with 1 n HCl to neutral pH, being sure not to overshoot to acid pH, because aminopterin is sensitive to acid pH.
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The concentration of the 100× stock is 0.04 mm aminopterin.
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3. Filter-sterilize the two solutions independently.
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4. Dispense 2.0-mL aliquots in sterile tubes. Store at −20°C.
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Both 100× HT and 100× A are stable for 1 yr at −20°C.
Data from Littlefield (1964).
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