1. LB Plates
2. LB Carb Plates
3. YPD Plates
4. SC (-) plates
5. 1x YPD
6. 1x LB
7. 2x LB Media
8. 3% Agar
9. 4% Agar
10. 20% Glucose
11. 20% Galactose
12. 10% Raffinose
13. 2x YEC
14. 2x YEP + trp
15. 2x SC –6
16. Drop-out Mix
17. 50 mM trp
18. 100 mM his
19. 100 mM leu
20. 100 mM lys
21. 20 mM ura
22. 16 mM ade
23. M9 Medium
24. 2x YNB
25. 5-FOA media
26. Top Agar
27. 2x VLP media (2x YNB/CAA)
28. Yeast media additives chart

(Luria Broth)

For Bacteria:

Ingredients

3% Agar

2x LB Broth

Directions

Loosen the cap on bottle of 3% agar and heat in microwave for 8 minutes at ½ power. After eight minutes, remove the bottle of agar and swirl slowly to melt the agar at the bottom of the bottle. When the bottle of agar is cool enough to touch, pour the contents into the 2x LB (the agar is more dense and will sink to the bottom). Mix well by swirling and inverting the bottle several times.

Pour the plates under the hood to lessen the growth of mold. After the agar has solidified, invert the plates and mark them with the appropriate code. Let the plates set out a few days to dry out and then place them in the cold room in the correct storage container.

***

Follow aseptic technique. This means flaming the lids of both bottles before pouring the agar into the broth. Also flame the lid often while pouring plates.

2. LB-Carb Plates

For growth and selection of bacteria with ampicillin resistant plasmid.

Ingredients:

3% Agar

2x LB Broth

500m l of 1000x carbenicillin (in freezer)

Directions:

Loosen cap on bottle of 3% agar and heat in microwave for 8 minutes at 1/2 power. After eight minutes, remove the bottle of agar and swirl slowly to melt the agar at the bottom of the bottle. Add the 500m l carbenicillin to the 2x LB broth. When the bottle of agar is cool enough to touch, pour the contents into the 2x LB (the agar is more dense and will sink to the bottom). Mix well by swirling and inverting the bottle several times.

Pour the plates under the hood to lessen the growth of mold. After the agar has solidified, invert the plates and mark them with the appropriate code (a black stripe). Let the plates set out a few days to dry out, and then place them in the cold room in the correct storage container.

***

Follow aseptic technique. This means flaming the lids of both bottles before pouring the agar into the broth. Also flame the lid often while pouring plates.

3. YPD Plates

(Yeast extract, Peptone, Dextose)

For yeast (S. cerevisiae).

Ingredients:

4% agar

2x YEP (yeast extract peptone) + trp

Dextrose (2% final)

Directions:

Loosen the cap on the bottle of 4% agar and heat in the microwave for 8 minutes at 1/2 power. After eight minutes, remove the bottle of agar and swirl slowly to melt the agar at the bottom of the bottle. Add 50ml dextrose to the YEP. When the bottle of agar is cool enough to touch, pour the contents into the 2x YEP (the agar is more dense and will sink to the bottom). Mix well by swirling and inverting the bottle several times.

Pour the plates under the hood to lessen the growth of mold. After the agar has solidified, invert the plates and mark them with the appropriate code ( a green stripe). Let the plates set out a few days to dry out and then place them in the cold room in the correct storage container.

***

Follow aseptic technique. This means flaming the lids of both bottles before pouring the agar into the broth. Also flame the lid often while pouring plates.

(Synthetic Complete)

2x SC –6 media 250ml

4% Agar 250ml

Leu 10ml

Ade 5ml

Lys 5ml

20% Glucose 50ml Carbon Sources

20% Galactose 50ml

Loosen cap and heat agar in microwave for for 8 minutes at 1/2 power. While heating the agar, add the desired carbon source (either Glucose or Galactose, but not both) and all amino acids except those that are to be lacking to the 2x SC –6. (For example, if the plates are to be –Ura, then all but Ura are added.) Swirl the agar to make sure that it is completely melted. Heat 2x SC –6 media for 1 minute at full power. Always add 4% agar to the 2x SC –6 (not the other way around). Add quickly, making sure not to pour agar onto the lid of the media bottle.

Obtain a stack of plates from the back room. Pour enough of the total mixture to cover the bottom of the plates. If bubbles form on the plates, use the burner to flame the surface of the media and the bubbles will disappear. Make the appropriate markings on the plates according to the preset marking system. Let plates set overnight. Before placing in the cold room, wipe the lids of the plates with a Kim-wipe with EtOH.

***

Follow aseptic technique. This means flaming the lids of both bottles before pouring the agar into the media. Also flame the lid often while pouring plates. Also, keep all bottles from the shelf on the lab bench because they may be contaminated after use. (Exception: Trp must go back into the refrigerator.)

(Yeast extract, Peptone, Dextose)

Broth for Yeast

Ingredients:

Yeast extract 5g

Bacto-peptone 10g

Glucose 10g

Distilled water 500ml

Directions:

Weigh out the correct amount of each ingredient in a separate weigh boat using an electric balance and transfer each to a flask. Add Milli-Q water and mix well using a spinbar. Distribute as 100ml into small bottles. Label with name and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

(Luria Broth)

Broth for bacteria.

Ingredients: 1x

Bacto-tryptone 5 g

Yeast Extract 2.5 g

Sodium chloride 5 g

Distilled H₂O 500ml

Directions:

Mix all of the ingredients together and add the water. Mix well by stirring, using a spinbar. After mixing, distribute as 100ml aliquots in small bottles. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle..

(Luria Broth)

Broth for bacteria.

Ingredients: 2x

Bacto-tryptone 20g

Yeast Extract 10g

Sodium chloride 20g

Distilled H₂O 1000ml

Directions:

Mix all of the ingredients together and add the water. Mix well by stirring, using a spinbar. After mixing, distribute as 250ml aliquots in 500ml bottles. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

For bacteria.

Ingredients:

DifcoAgar 7.5g

Distilled H₂O 250ml

Directions:

Weigh out 7.5g of agar into a 500ml bottle. Add the water. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

For yeast

Ingredients:

DifcoAgar 10g

Distilled H₂O 250ml

Directions:

Weigh out 10g of agar into a 500ml bottle. Add the water. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Media supplement for bacteria, yeast, and pombe.

Ingredients:

Dextrose (D-glucose) 20g 100g

Distilled H₂O 100ml 500ml

Directions:

Add approximately 4/5 the volume of distilled water (80ml or 400ml, respectively for above volumes) to a flask. Place a spinbar in the flask and begin mixing. Add the proper amount of glucose to the flask, heating slightly to assure the glucose dissolves. When dissolved, pour the solution back into a graduated cylinder and bring up to total volume (100ml or 500ml). Mix well before distributing as 100ml aliquots in 220cc bottles (flattened sides). Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Media supplement for bacteria, yeast, and pombe.

Ingredients:

D-galactose 20g 100g

Distilled H₂O 100ml 500ml

Directions:

Weigh out 20g of galactose into a flask. Mix the galactose in 4/5 of the volume of distilled water (80ml or 400ml, respectively for above volumes) using a spinbar. When dissolved, pour solution back into a graduated cylinder and bring up to total volume (100ml or 500ml). Mix well and distribute as 100ml aliquots in 220cc bottles (flattened sides). Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Ingredients:

Raffinose 10g 50g

Distilled H₂O 100ml 500ml

Directions:

Weigh out 10g of raffinose into a flask. Mix the raffinose in 4/5 of the volume of distilled water (80ml or 400ml, respectively for above volumes) using a spinbar. When dissolved, pour solution back into a graduated cylinder and bring up to total volume (100ml or 500ml). Mix well and distribute as 100ml aliquots in 220cc bottles (flattened sides). Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

(Yeast Extract Casamino acids)

Broth for pombe.

Ingredients:

Yeast extract 5g

Casamino acids 2g

Distilled H₂O 500ml

Directions:

Weigh out 5g of yeast extract and transfer it to a 1000ml flask. Add 2g of casamino acids. Add the distilled water and mix, using a spinbar. Distribute as 250ml in two bottles. Label the bottles with name and concentration. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Note: Usually make up a liter and distribute into four bottles.

(Yeast Extract Peptone)

Broth for yeast.

Ingredients:

Yeast extract (Bacto) 20g

Bacto-peptone 40g

Tryptophan 0.64g

Distilled H₂O 1000ml

Directions:

Mix all the ingredients together and add the distilled water. Mix well using a spinbar. Distribute as 250ml into 500ml bottles. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

(Synthetic Complete)

Ingredients:

Yeast nitrogen base 9g

Ammonium sulfate 25g

"-6" amino acid mix 10g

Milli-Q water 2.5L

Directions:

Weigh out the correct amount of each ingredient in a separate weigh-boat using an electric balance and transfer each to a flask. Add Milli-Q water and mix to dissolve using a spinbar. Distribute as 250ml into 10 500ml Wheaton bottles. Label with name and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Amino acid supplement for yeast, pombe.

L-Tryptophan 2g

Milli-Q water 250ml

Weigh out the correct amount of tryptophan in a weigh-boat using an electric balance and transfer to a flask. Add Milli-Q water and mix to dissolve using a spinbar. Distribute as 50ml into 5 bottles. Label with name, concentration and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle. Store at 4° C, wrapped in foil.

Amino acid supplement for yeast, pombe.

L-Histidine HCl*H₂O 2.1g

Milli-Q water 100ml

Weigh out the correct amount of histidine in a weigh-boat using an electric balance and transfer to a flask. Add Milli-Q water and mix to dissolve using a spinbar. Distribute as 50ml into 2 bottles. Label with name, concentration and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Amino acid supplement for yeast, pombe.

L-Leucine 1.31g 1.96g

Milli-Q water 100ml 150ml

Weigh out the correct amount of Leucine in a weigh-boat using an electric balance. Cover this with Milli-Q water. Transfer to a flask and fill to the desired volume with Milli-Q water. Mix to dissolve using a spinbar. Distribute as 50ml into the appropriate number of bottles. Label with name, concentration and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Amino acid supplement for yeast, pombe.

L-Lysine (mono-HCl) 1.83g 2.75g

Milli-Q water 100ml 150ml

Weigh out the correct amount of lysine in a weigh-boat using an electric balance and transfer to a flask. Add Milli-Q water and mix to dissolve using a spinbar. Distribute as 50ml into the appropriate number of bottles. Label with name, concentration and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Amino acid supplement for yeast, pombe.

Uracil 0.224g 0.336g

Milli-Q water 100ml 150ml

Weigh out the correct amount of uracil in a weigh-boat using an electric balance and transfer to a flask. Add Milli-Q water and mix to dissolve using a spinbar. Distribute as 50ml into the appropriate number of bottles. Label with name, concentration and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Amino acid supplement for yeast, pombe.

Adenine hemisulfate 0.3g 0.45g

Milli-Q water 100ml 150ml

Weigh out the correct amount of adenine hemisulfate in a weigh-boat using an electric balance and transfer to a flask. Add Milli-Q water and mix to dissolve using a spinbar. Distribute as 50ml into the appropriate number of bottles. Label with name, concentration and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

(Minimal (for bacteria))

Sodium Phosphate Dibasic (Na₂HPO_4* 7H₂O) 25.6g

Potassium Phosphate Dibasic (KH₂PO₄) 6.0g

Sodium Chloride (NaCl) 1.0g

Ammonium Chloride (NH₄Cl) 2.0g

Weigh out the correct amount of each ingredient in a separate weigh-boat using an electric balance and transfer each to a flask. Add Milli-Q water and mix to dissolve using a spinbar. Distribute as 250ml into 4 500ml Wheaton bottles. Label with name and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

1M MgSO₄ 1ml

20% Glucose 5ml

50 mM CaCl₂ 1ml

0.2% Thaimine 0.5ml

Add the above to 2x M9 (250ml) along with:

For plates: 250ml 3% agar

For broth: 250ml sterile water

Other additions:

10% CAA (cas amino acids) 10ml

20 mM Uracil 5ml

40 mM Tryptophan 5ml

For –Ura For -Leu

10% CAA 10ml 40 mM Ura 5ml

40 mM Trp 5ml 40 mM Trp 5ml

(Yeast Nitrogen Base)

3.6 g Yeast Nitrogen Base

10 g Ammonium sulfate

1000 ml water

Directions:

Weigh out the correct amount of each ingredient in a separate weigh-boat using an electric balance and transfer each to a flask. Add Milli-Q water and mix to dissolve using a spinbar. Distribute as 250ml into 4 500ml Wheaton bottles. Label with name and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

25. 5-FOA plates

(5-Fluoroorotic Acid)

250 ml 2X SC-6

0.5 g 5-FOA (5-flouroorotic acid)

25 mg uracil

50 ml 20% glucose

250 ml 4% agar

Weigh the 5-FOA and uracil into the SC-6. Heat in microwave 1 or 2 minutes and swirl until all powder is dissolved.

Loosen cap and heat agar in microwave for 8 minutes at 1/2 power. While heating the agar, add the glucose and all amino acids except those that are to be lacking and URACIL to the 2x SC –6. (For example, if the plates are to be FOA-His, then all but His are added.) Swirl the agar to make sure that it is completely melted. Heat 2x SC –6 media for 1 minute at full power. Always add 4% agar to the 2x SC –6 (not the other way around). Add quickly, making sure not to pour agar onto the lid of the media bottle.

Obtain a stack of plates from the back room. Pour enough of the total mixture to cover the bottom of the plates. If bubbles form on the plates, use the burner to flame the surface of the media and the bubbles will disappear. Make the appropriate markings on the plates according to the preset marking system. Let plates set overnight. Before placing in the cold room, wipe the lids of the plates with a Kim-wipe with EtOH.

For phage infection.

Per 250ml:

Bactotryptone 2.5g

Yeast extract 1.3g

NaCl 1.3g

Agar 1.8g

Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

Media for isolating Virus-Like Particles

3.6 g YNB

10 g Ammonium sulfate

40 g Cas Amino Acids

1000 ml water

Directions:

Weigh out the correct amount of each ingredient in a separate weigh-boat using an electric balance and transfer each to a flask. Add Milli-Q water and mix to dissolve using a spinbar. Distribute as 250ml into 4 500ml Wheaton bottles. Label with name and date. Cap loosely, pre-warm, and autoclave for 20 minutes, liquid cycle.

For your convenience, the chart below can be cut out and taped up at your lab bench.