Protocols for Chlamy

Tris　             2.42g

Hutner’s Trace element* 1.0ml

CH3COOH                1.0ml

10% CaCl2・2H2O        0.5ml

20% NH4Cl              2.0ml

30% MgSo4・7H2O        1.0ml

10% KH2・PO4           1.0ml

10% K2H・PO4           1.0ml
—————————————————————————————————
total                 1 L

＊To prepare agar plates, add 1.2%~1.5%.

＊minimum medium (lacking the carbon source)

Omit CH3COOH 1.0ml and adjust pH to 7.0 with HCl ~1.6ml/l.

• • It will take 10 days to be completed. Prepare early. The preparation should be done in the hume hood.
    
    1.Dissolve EDTA（acid free）in Milli Q 250ml.
    
    Heat the solution to ~100 ºC (A)
    
    2.Heat MilliQ 550ml to ~100℃ and add the reagents as follows in order (B)
    
    H3 BO3           11.4g
    
    ZnSO4・7H2O      22.0g
    
    MnCl2・4H2O      5.06g
    
    FeSO4・7H2O      4.99g
    
    CoCl2・6H2O      1.61g
    
    CuSO4・5H2O      1.57g
    
    Mo7O24(NH4)6・4H2O 1.10g (Ammonium Molybdate)
    
    

    1. Mix (A) and (B) at ~100ºC. If successful, the solution will be “creepy” blue green.

    2. Cool down the solution to 80〜90℃. Adjust pH to 6.5〜6.8 with KOH. Keep the solution warmer than 70ºC.

  • （~83 mL of 20% KOH may be needed）
    
    

    1. Add DW to make the solution 1L, then transfer the solution to 2L flask and let it cool down in room temp. (Be careful with the hot solution!)

    2. After cooling down, seal the flask with a parafilm and leave it at room temperature for 10 days.

  • 7.If the color changes from bluish green to transparent purple, SUCCESS! Filter the debris and use the supernatant as Hutner’s trace element.
    
    

10X Tsubo mating buffer (Stock solution)

500 mM HEPES / KOH (pH 6.8)  12 mL

500 mM MgSO4            10 mL

-------------------------------------------------------------

total 500 mL

Replace 30% MgSO4・7H2O 1.0ml with 30% MgCl2・7H2O 1.0ml.

Omit CH3COOH and adjust the pH with HCl to ~7.0

For gametogenesis. You usually do not have to adjust pH （〜7.0）

5% Na Citrate・2H2O    10.0ml

20% CH3COONa           10.0ml

1% FeCl3・6H2O         1.0ml

Trace element     1.0ml

4% CaCl2・2H2O         1.0ml

30% MgSO4・7H2O        1.0ml

30% NH4NO3             1.0ml

10% K2HPO4 / 10% KH2PO4   1.0ml

—————————————————————————————————

total                 1 L

＊Trace element（SGⅡ）

Mix the reagents and filtrate

ZnSO4・7H2O            0.30g

H3BO3                 0.30g

MnCl2・4H2O            0.12g（MnSO4・4H2O 0.12g is also OK）

CoCl2・6H2O            0.06g

NaMoO4・2H2O           0.06g

CuSO4                  0.012g

—————————————————————————————————

total                 300 ml

Chlamydomonas electroporation with NEPA21

Reagents:

TAP-60mM sucrose

Add sucrose to liquid TAP medium and sterilize with filtration.

CHES buffer

10 mM N-cyclohexyl-2-aminoethane sulfonic acid (CHES) pH 9.25

40 mM sucrose

10 mM sorbitol

 Sterilize with filtration

1) Prepare cell cultures at the log phase ~106 cells/mL

2) 2000 rpm for 3 min at 4ºC

3) Discard the supernatant

4) Suspend the cells in TAP-60 mM sucrose (or sorbitol)

5) 2000 rpm for 2 min at 4ºC

6) Discard the supernatant

7) Add CHES buffer to adjust the cell concentration to ~108 cells/mL

8) Mix 1.5 µL of DNA (1 µg/mL) and 50 µL of cell suspension on ice.

9) Prepare cuvettes (2 mm gap) and add ~40 µL of the mixture to the cuvette (no bubbles!)

10) Set the cuvette in NEPA21

11) Push “Ω” and record the impedance before the electroporation

12) Set the parameters for electroporation

13) Prepare 8 mL TAP-60mM sucrose in 15 mL tubes.

14) Fire!

15) After the electroporation, transfer the cells to 15 mL tubes and incubate them for overnight at room temperature under dim light or in the dark.

16) 2000 rpm for 5 min

17) Spread the cells onto TAP medium containing appropriate antibiotics.

Parameters

Poring pulse: 250V, Pulse length: 8 msec, Pulse interval: 50 msec, Pulses: 2, Decay rate: 40%, Polarity: +

Transfer pulse: 20V, Pulse length: 50 msec, Pulse interval: 50 msec, Pulses: 5, Decay rate: 40%, Polarity: +/-

*For CC-125 mt+, the poring pulse should be 300V.