NGM plates
*makes 1L*
17g BactoAgar
2.5g peptone
3g NaCl
Add 975mL of dH2O and add magnetic stir bar
Autoclave on Liquid30 cycle (1 hour)
Allow to cool for 15 minutes while stirring, then add:
25mL 1M KPO4
1mL 1M CaCl2
1mL 1M MgSO4
1mL cholesterol
Dry overnight while bacteria shake
Seed NGM plates with OP50-1
In Falcon 50 tube, pour 15mL of LB media
Pipette 1µL/mL (15µL) of streptomycin antibiotic into Falcon 50 tube
Use a pipette tip to stab bacterial strain glycerol stock found in -80ºC freezer
Incubate in shaking incubator for 24 hours
Spin down OP50-1 for 10 minutes at 4000rpm
Discard supernatant and resuspend pellet in half volume of M9 (for 15mL, resuspend in 7.5mL M9)
To seed NGM plates:
Small plate: 30µL
Medium plate: 100µL
Large plate: 200µL
Note: if seeding with OP50, no antibiotic necessary
Let plates dry overnight, then move plates to containers and store in 4ºC fridge
Always move plates after drying overnight, if left at room temperature, contamination will grow
IPTG Plates (RNAi plates)
After cooling step, add all salts and reagents to cooled NGM.
Add 1mM IPTG (0.2383g) directly to NGM
IPTG is found in large 4ºC fridge
Best to measure out 1mM IPTG in Eppendorf tube before adding it to NGM bottle
Optional: add ampicillin to 50µg/mL (current ampicillin stocks are 100mg/mL, add 500µL of ampicillin stock)
Dry plates for 4-7 days before seeding
1M KPO4
*Makes 500mL*
Add 54.15g 〖KH2 PO]4 (monobasic) to 300mL dH2O
Add 17.8g 〖K_2 HPO]4 (dibasic)
Fill to 500mL with dH2O
1M CaCl2
*makes 500mL*
Add 55.45g CaCl2 to 250mL dH2O
Fill to 500mL total volume
Stir to dissolve
1M MgSO4
*makes 500mL*
Add 60.15g MgSO4 into 250mL of dH2O
Fill to 500mL total volume
Stir to dissolve
Cholesterol stocks
Add 0.25g cholesterol (found in -20ºC freezer) to 50mL of ethanol in Falcon 50 tube
Concentration of 5mg/mL
Stir on rotisserie until cholesterol has completely dissolved
Aliquot 1mL into Eppendorf tubes and store in -20ºC freezer
M9 Worm Media
*makes 500mL*
Add 1.5g KH2PO4, 3g Na2HPO4, 2.5g NaCl, and dH2O up to 500mL
Autoclave on liquid 30 cycle
Add 1mL 1M MgSO4 after liquid has cooled
Store in ambient fridge at room temperature
Worm Freezing Solution
Prepare S buffer:
129 ml 0.05 M K2HPO4
871 ml 0.05 M KH2PO4
5.85g NaCl
Autoclave 30% glycerol solution
Combine S buffer and 30% glycerol solution in 1:1 ratio
LB Media
*makes 1L*
10g BactoTryptone
5g Yeast extract
10g NaCl
Add dH2O to 1000mL total
Autoclave liquid30 cycle
Antibiotic plates
*makes 800mL*
8g BactoTryptone
4g Yeast extract
8g NaCl
12g BactoAgar
Add dH2O to 800mL total
Autoclave on Liquid30 cycle
Cool while stirring for 45 minutes
Add 800uL of thawed ampicillin stock (plate concentration 100µg/mL) or kanamycin stock (plate concentration 50µg/mL)
Antibiotic stock solutions
For additional antibiotic stock and plate information, see At the Bench, a Laboratory Navigator p. 258
Ampicillin
*100mg/mL*
1g Ampicillin sodium salt (A9518-25G)
10mL nuclease free dH2O
Filter to sterilize
Aliquot to 1mL stocks
Kanamycin
*50mg/mL*
0.5g Kanamycin sulfate from Streptomycin kanamyceticus (K1377-5G)
10mL nuclease free dH2O
Filter to sterilize
Aliquot to 1mL stocks
Streptomycin
*100mg/mL*
1g Streptomycin sulfate salt (S6501-100G)
10mL nuclease free dH2O
Filter to sterilize
Aliquot to 1mL stocks
Chloramphenicol
*20mg/mL*
0.2g Chloramphenicol (C0378-5G)
10mL ethanol (under fume hood)
Aliquot to 1mL stocks
Tetracycline
*15mg/mL*
0.15g tetracycline (87128-100G)
5mL ethanol (under fume hood) and 5mL nuclease free dH2O
Aliquot to 1mL stocks
Note: this is light and temperature sensitive, if stock is discolored (appears red instead of usual rust color) then throw away
SOB
*makes 1L*
20g BactoTryptone
5g Yeast extract
0.5g NaCl
Add 10mL of 250mM KCl
Add dH2O to 1000mL total and shake to dissolve
Add stir bar, allow to stir until all salt appears to have dissolved
Adjust to pH 7 with 0.2mL 1M NaOH (do in 100µL increments)
Autoclave on Liquid30 cycle
Cool, add 5mL of 2M MgCl2
Transformation Buffer
3.0g PIPES (Sigma P-9291)
2.2g CaCl2-2H2O (Sigma C-7902)
18.6g KCl (Sigma P-0217)
Dissolve in 975ml ultrapure water (Aldrich, Cat#32007-2; milliQ and ddH2O are also good, but make sure the filters are working right).
Add stir bar and stir to dissolve salts.
Bring pH up to 6.7-6.8 by adding drops of 5 N KOH (white precipitates will not go into solution until the pH is close to 6.0).
Add 10.9g MnCl2-4H2O
Add water up to 1L, filter with 0.22μm sterile filter unit and keep it at 4℃ (lasts for several months).
DH5α Competent Cells
Protocol for making DH5α competent cells
Inoculate 5ml LB liquid media with DH5α single colony or glycerol stock (5μl) and incubate on shaker (~300rpm) overnight at 37℃.
Inoculate 5ml DH5α into 500ml SOB in 2L flask and incubate on shaker (~300rpm) at 20℃.
Incubate until the culture density reaches between 0.4~0.6 OD at 600nm. (It usually takes ~25-30hr at 18℃). If flask is put in shaker at approximately 4:00pm, OD should be checked throughout the following morning.
Split into two 250ml centrifuge bottles.
**Steps 5 to 11 need to be done inside the COLD ROOM (4℃)**
Spin cells down at 3000rpm for 10min at 4℃
Take off the supernatant and add 1/3 volume (80ml) of ice-cold Transformation buffer (TB).
Resuspend the pellet GENTLY using a 25ml pipette and transfer to Falcon 50 tubes and leave it on ice for 10min.
Spin cells down as above.
Take off the supernatant and add 1/12.5 volume (4ml) of ice-cold TB.
Resuspend the pellet GENTLY using a 25ml pipette.
Add DMSO to 7% (v/v) (280µl) and transfer to flask and leave cells on ice for 10min.
Aliquot them into pre-chilled 96-well PCR plates or vials (50μl/well in 96-well). Flask must be kept on ice and gently swirled.
Freeze them immediately in liquid nitrogen or on dry ice and store frozen at -80℃.
1M Glucose
100mL dH2O
18g glucose
Filter to sterilize
SOC
*makes 1L*
950mL〖H2O]
20g BactoTryptone
5g Yeast
0.5g NaCl
Fill to 1L total volume
Autoclave Liquid30 and allow to cool
Cool, add 5mL of 2M MgCl2
Add 20mL 1M glucose
50X TAE
*makes 1L*
600mL milliQH2O
57mL acetic acid
100mL 0.5M EDTA
242g Tris
Fill to 1L
Note: to dilute to 1X TAE, combine 20mL 50X TAE with 980mL dH2O
0.5M EDTA
250mL dH2O
46.53g EDTA
Add stir bar
Place on stir plate and add 45g NaOH pellets to dissolve
TB Broth
*makes 1L*
900mL dH2O
12g BactoTryptone
24g Yeast
4mL glycerol
Do not adjust to 1L
Autoclave Liquid30
Allow to cool and add 100mL potassium stock
Potassium
*stocks for TB broth – makes 250mL*
225mL dH2O
5.78g KH2PO4 (monobasic)
31.35g K2HPO4 (dibasic)
Water up to 250mL total volume
Autoclave Liquid30
