Day 1

YAPD = YPD + adenine (80 mg/L). Adenine can be added to the media before autoclaving

-leu + adenine + amp plates (0.5 L):

• Yeast synthetic drop-out medium
• Supplement w/o leucine (BIO 101 CSM-leu) 0.8 g
• Yeast nitrogen base w/o amino acids 3.33 g
• Glucose 10 g
• Adenine 40 mg
• Difco agar 10 g

Autoclave, cool and add amp before pouring.

Expected transformation results

Plasmids

Day 2

YAPD plates = YPD agar + adenine (80 mg/L). Adenine can be added to media before autoclaving

Autoclave, cool to 50°C and add nourseothricin (50 μg /ml) and/or amp before pouring

-met + adenine + amp plates (0.5 L):

• Yeast synthetic drop-out medium supplement w/o methionine (BIO 101 CSM-leu): 0.8 g
• Yeast nitrogen base w/o amino acids: 3.33 g
• Glucose: 10 g
• Adenine: 40 mg
• Difco agar: 10 g

Autoclave, cool and add amp before pouring.

Fake -leu + adenine + amp plates (for transformation rescue plates)

(i.e. as per recipe for -leu plates in CRISPR #1 but with drop-out without methionine and without leucine, so not actually selective) (0.5 L):

• Yeast synthetic drop-out medium supplement w/o methionine: 0.4 g
• Yeast synthetic drop-out medium supplement w/o leucine: 0.4 g
• Yeast nitrogen base w/o amino acids: 3.33 g
• Glucose: 10 g
• Adenine: 40 mg
• Difco agar: 10 g

Notes on checking growth of S. cerevisiae K699

Need for adenine

K699 is apparently an auxotroph for adenine. Check how presence of 40 mg/L adenine affects growth on complex media (YPD). NB adenine-HCl added before autoclaving, as I had trouble making up a stock solution of adenine-HCl (would not dissolve in water, even at half the concentration of Sigma’s stated solubility).

• Grew K699 O/N at 30°C/shaking in 5 ml YPD or YAPD, from a glycerol stock or a plate inoculation (both cultures grew).
• Then streaked all four cultures onto YPD + amp plates or YAPD + amp plates
• Incubated at 30°C for 2d

Appears that K699 can grow on YPD without adenine, but that the addition of adenine (RH plates) results in bigger colonies.  So continue to add approx. 40 mg/L adenine-HCl to YPD media for growth of this strain.

Update: after a few days storage in fridge, cultures grown on YPD plates were distinctly red

OD for transformations

Protocol states that K699 should be at OD600 nm ~0.4-2.0.  Test OD of overnight culture vs A16 (which also has to be in log phase for mol gen transformations in autumn).

A16 is grown as follows in autumn: 200 μl inoculum into 350 ml YPD in 2 L flask, grown at 30°C overnight at 200 rpm.

Scale down to 44 ml in 250 ml flask with 25 μl inoculum, grown at 30°C overnight at 200 rpm.  Grow A16 in YPD and K699 in YAPD

OD_600nm after 16 h:

A16 = 1.17

K699 = 1.06

So K699 grows to a similar OD_600nm as A16, suggesting it should be OK (assuming Hansenula and Saccharomyces behave similarly in transformations). Make a note to take the flask out on the morning of the class and put on ice to avoid any further growth.

Nourseothricin concentration

Protocols state to use 50-100 μg/ml nourseothricin.  Check growth of K699 (bottom row) vs a control NatMx+ strain (top row) on these concentrations:

0                                  50                                  100 ug/ml

50 μg/ml is sufficient, 100 μg/ml is not detrimental to growth

Access to plasmids

Please contact Mel Stapleton (m.stapleton@sheffield.ac.uk) to make arrangements.