YAC TRANSFER by KAR1

YAC transfer by Kar1 mating:

Reference:

Hugerat, Y., Spencer, F., Zenvirth, D. and Simchen, G. (1994). A versatile method for efficient YAC transfer between any two strains. Genomics 22, 108-117
2. Spencer, F., Hugerat, Y., Simchen, G., Hurko, O., Connelly, C. and Hieter, P. (1994). Yeast kar1 mutants provide an effective method for YAC transfer to new hosts. Genomics 22, 118-126


Transfer YAC from donor cells into YPH 925

 

1. YAC strain ( MAT a) is grown in selective medium and YPH925 ( MAT alpha) was grown in liquid YPD medium until late log phase.
2. For matings, 5X10 ⁶ cells of each of the two parent strains are combined in liquid YPD medium and incubated for 6-8 hours or longer at 23ºC (with or without stirring).
3. Cells are collected by centrifugation and washed twice with sterile water, and then cells are plated at about 5X10 ⁶ cells per plate on cycloheximide medium ( 3.0 µg/ml cycloheximide) selective for the YAC transfer.
4. YACductants can be distinguished from the other products by one recessive marker carried by YPH925 or by mating type test.
5. YAC transfer can be confirmed by the appearance of a DNA band of the expected size in pulsed-field gel electrophoresis of chromosomal DNA from the YACductant.

Transfer YAC from YPH925 into recipient cells

1. Recipient strain ( MAT a and canavanine resistant) and YPH925 ( MAT alpha) is grown in liquid YPD medium until late log phase.
   
2. For matings, 5X10 ⁶ cells of each of the two parent strains are combined in liquid YPD medium and incubated for 6-8 hours at 23ºC.
   
3. Cells are collected by centrifugation and washed twice with sterile water, and then cells are plated at about 5X10 ⁶ cells per plate on canavanine medium ( SC medium lacking arginine and supplemented with 40 µg/ml canavanine) selective for the YAC transfer.
   
4. YACductants can be distinguished from the other products by one recessive marker carried by recipient cells or by mating type test.
   
5. YAC transfer can be confirmed by the appearance of a DNA band of the expected size in pulsed-field gel electrophoresis of chromosomal DNA from the YACductant.

Comments: Expected number of transductants is 50-200/plate. Background of other events is about 5-10/plate

<center> <p>  </p> </center>
上一篇：Sending Strains   下一篇：Western blotting using ECL reagent