Griffitts:Stock solutions: Difference between revisions
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==Antibiotics== | ==Antibiotics== | ||
===Ampicillin (40 mL)=== | ===Ampicillin (40 mL)=== | ||
For a stock solution of 25 mg/mL: | |||
* 1 g [[Wikipedia:Ampicillin|Ampicillin]] | * 1 g [[Wikipedia:Ampicillin|Ampicillin]] | ||
* 40 mL ddH<sub>2</sub>O | * 40 mL ddH<sub>2</sub>O | ||
* Filter sterilize | * Filter sterilize | ||
NOTE: Store at 4°C | |||
===Chloramphenicol (40 mL)=== | ===Chloramphenicol (40 mL)=== | ||
* | For a stock solution of 7.5 mg/mL: | ||
* 300 mg [[Wikipedia:Chloramphenicol|Chloramphenicol]] | |||
* 40 mL 50% [[Wikipedia:Ethanol|Ethanol]] | * 40 mL 50% [[Wikipedia:Ethanol|Ethanol]] | ||
NOTE: Do '''not''' filter sterilize—the ethanol may dissolve the filter<br> | |||
NOTE: Store at 4°C | |||
===Erythromycin (20 mL)=== | ===Erythromycin (20 mL)=== | ||
* | For a stock solution of 10 mg/mL: | ||
* 200 mg [[Wikipedia:Erythromycin|Erythromycin]] | |||
* 20 mL 100% ethanol (in fume hood) | * 20 mL 100% ethanol (in fume hood) | ||
* Do '''not''' filter sterilize—the ethanol may dissolve the filter | * Do '''not''' filter sterilize—the ethanol may dissolve the filter | ||
** Store | NOTE: Store at –20°C | ||
===Gentamicin (20 mL)=== | |||
For a stock solution of 7.5 mg/mL: | |||
* 150 mg [[Wikipedia:Gentamicin|Gentamicin]] | |||
* 20 mL ddH<sub>2</sub>O | |||
* Filter sterilize | |||
NOTE: Store at 4°C | |||
=== | ===Hygromycin (40 mL)=== | ||
* | For a stock solution of 5 mg/mL: | ||
* 200 mg [[Wikipedia:Hygromycin|Hygromycin]] | |||
* 40 mL ddH<sub>2</sub>O | * 40 mL ddH<sub>2</sub>O | ||
* Filter sterilize | * Filter sterilize | ||
NOTE: Store at 4°C | |||
===Kanamycin (40 mL)=== | ===Kanamycin (40 mL)=== | ||
* | For a stock solution of 7.5 mg/mL: | ||
* 300 mg [[Wikipedia:Kanamycin|Kanamycin]] | |||
* 40 mL ddH<sub>2</sub>O | * 40 mL ddH<sub>2</sub>O | ||
* Filter sterilize | * Filter sterilize | ||
NOTE: Store at 4°C | |||
===Nalidixic acid (30 mL)=== | ===Nalidixic acid (30 mL)=== | ||
* | For a stock solution of 30 mg/mL: | ||
* | * 900 mg [[Wikipedia:Nalidixic Acid|Nalidixic Acid]] | ||
* 360 mg NaOH | |||
* 30 mL ddH<sub>2</sub>O | * 30 mL ddH<sub>2</sub>O | ||
* Filter sterilize | * Filter sterilize | ||
NOTE: Store at 4°C | |||
===Neomycin (40 mL)=== | ===Neomycin (40 mL)=== | ||
* | For a stock solution of 25 mg/mL: | ||
* 1 g [[Wikipedia:Neomycin|Neomycin]] | |||
* 40 mL ddH<sub>2</sub>O | * 40 mL ddH<sub>2</sub>O | ||
* Filter sterilize | * Filter sterilize | ||
** Store | NOTE: Store at 4°C | ||
===Phosphomycin=== | |||
For a stock solution of 25 mg/mL: | |||
* 1 g [[Wikipedia:Fosfomycin|Phosphomycin]] | |||
* 40 mL ddH<sub>2</sub>O | |||
* Filter sterilize | |||
NOTE: Store at 4°C | |||
===Rifampicin (20 mL)=== | ===Rifampicin (20 mL)=== | ||
* 1 | For a stock solution of 50 mg/mL: | ||
* 1 g [[Wikipedia:Rifampicin|Rifampicin]] | |||
* 20 mL N,N-dimethylformamide (DMF; in fume hood) | * 20 mL N,N-dimethylformamide (DMF; in fume hood) | ||
NOTE: Do '''not''' filter sterilize—the DMF will dissolve the filter<br> | |||
NOTE: Store at –20°C | |||
===Spectinomycin (40 mL)=== | ===Spectinomycin (40 mL)=== | ||
* | For a stock solution of 20 mg/mL: | ||
* 800 mg [[Wikipedia:Spectinomycin|Spectinomycin]] | |||
* 40 mL ddH<sub>2</sub>O | * 40 mL ddH<sub>2</sub>O | ||
* Filter sterilize | * Filter sterilize | ||
NOTE: Store at 4°C | |||
===Streptomycin (40 mL)=== | ===Streptomycin (40 mL)=== | ||
* 2 | For a stock solution of 50 mg/mL: | ||
* 2 g [[Wikipedia:Streptomycin|Streptomycin]] | |||
* 40 mL ddH<sub>2</sub>O | * 40 mL ddH<sub>2</sub>O | ||
* Filter sterilize | * Filter sterilize | ||
NOTE: Store at 4°C | |||
===Tetracycline (40 mL)=== | ===Tetracycline (40 mL)=== | ||
* | For a stock solution of 2.5 mg/mL: | ||
* 100 mg [[Wikipedia:Tetracycline|Tetracycline]] | |||
* 40 mL 50% [[Wikipedia:Ethanol|Ethanol]] | * 40 mL 50% [[Wikipedia:Ethanol|Ethanol]] | ||
** Do '''not''' filter sterilize | NOTE: Do '''not''' filter sterilize—the ethanol may dissolve the filter<br> | ||
** Store | NOTE: Store at 4°C | ||
===Triclosan (40 mL)=== | |||
For a stock solution of 10 mg/mL: | |||
* 400 mg [[Wikipedia:Triclosan|Triclosan]] | |||
* 40 mL 100% [[Wikipedia:Ethanol|Ethanol]] | |||
NOTE: Do '''not''' filter sterilize—the ethanol may dissolve the filter<br> | |||
NOTE: Store at 4°C | |||
===Trimethoprim (40 mL)=== | |||
For a stock solution of 6.25 mg/mL: | |||
* 250 mg [[Wikipedia:Trimethoprim|Trimethoprim]] | |||
* 40 mL 100% [[Wikipedia:Dimethyl sulfoxide|DMSO]] | |||
NOTE: Do '''not''' filter sterilize—the DMSO may dissolve the filter<br> | |||
NOTE: Store at 4°C | |||
==Antibiotics Table== | ==Antibiotics Table== | ||
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! width="120" style="background:#efefef;" | ''E. coli'' | ! width="120" style="background:#efefef;" | ''E. coli'' | ||
! width="120" style="background:#efefef;" | ''S. meliloti'' | ! width="120" style="background:#efefef;" | ''S. meliloti'' | ||
! width=" | ! width="90" style="background:#efefef;" | Solvent | ||
! width=" | ! width="90" style="background:#efefef;" | Storage | ||
|- | |- | ||
| Ampicillin | | Ampicillin | ||
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| 30 μg/mL (250X) | | 30 μg/mL (250X) | ||
| N/A | | N/A | ||
| EtOH | | 50% EtOH | ||
| 4°C | | 4°C | ||
|- | |- | ||
| Erythromycin | | Erythromycin | ||
| | | Er | ||
| (none) | | <center>(none)</center> | ||
| | | 10 mg/mL | ||
| N/A | | N/A | ||
| N/A | | N/A | ||
| EtOH | | 100% EtOH | ||
| –20°C | | –20°C | ||
|- | |- | ||
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| Hygromycin | | Hygromycin | ||
| Hy | | Hy | ||
| (none) | | <center>(none)</center> | ||
| 5 mg/mL | | 5 mg/mL | ||
| 20 μg/mL (250X) | | 20 μg/mL (250X) | ||
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| 7.5 mg/mL | | 7.5 mg/mL | ||
| 30 μg/mL (250X) | | 30 μg/mL (250X) | ||
| | | '''Use Neomycin''' | ||
| ddH<sub>2</sub>O | | ddH<sub>2</sub>O | ||
| 4°C | | 4°C | ||
Line 137: | Line 167: | ||
| Nalidixic acid | | Nalidixic acid | ||
| Na | | Na | ||
| (none) | | <center>(none)</center> | ||
| | | 30 mg/mL | ||
| 30 μg/mL (1000X) | | 30 μg/mL (1000X) | ||
| N/A | | N/A | ||
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| Nm | | Nm | ||
! style="background:#008000;" | <font color=white>'''GREEN'''</font> | ! style="background:#008000;" | <font color=white>'''GREEN'''</font> | ||
| | | 25 mg/mL | ||
| '''Use Kanamycin''' | |||
| 100 μg/mL (250X) | |||
| ddH<sub>2</sub>O | |||
| 4°C | |||
|- | |||
| Phosphomycin | |||
| Pm | |||
| <center>(none)</center> | |||
| 25 mg/mL | |||
| N/A | | N/A | ||
| | | 100 μg/mL (250X) | ||
| ddH<sub>2</sub>O | | ddH<sub>2</sub>O | ||
| 4°C | | 4°C | ||
|- | |- | ||
| Rifampicin | | Rifampicin | ||
| | | Rf | ||
| (none) | | <center>(none)</center> | ||
| 50 mg/mL | | 50 mg/mL | ||
| N/A | | N/A | ||
| | | 100 μg/mL (500X) | ||
| DMF | | DMF | ||
| –20°C | | –20°C | ||
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! style="background:#FF0000;" | <font color=white>'''RED'''</font> | ! style="background:#FF0000;" | <font color=white>'''RED'''</font> | ||
| 2.5 mg/mL | | 2.5 mg/mL | ||
| | | 5 μg/mL (500X) | ||
| | | 5 μg/mL (500X) | ||
| EtOH | | 50% EtOH | ||
| 4°C | | 4°C | ||
|- | |- | ||
| Triclosan | | Triclosan | ||
| | | Tr | ||
| (none) | | <center>(none)</center> | ||
| | | 10 mg/mL | ||
| N/A | |||
| N/A | | N/A | ||
| 100% EtOH | |||
| 4°C | |||
|- | |||
| Trimethoprim | |||
| Tm | |||
| <center>(none)</center> | |||
| 6.25 mg/mL | |||
| N/A | | N/A | ||
| | | 12.5 μg/mL (500X)<br><center>or</center>25 μg/mL (250X) | ||
| 100% DMSO | |||
| 4°C | | 4°C | ||
|} | |} | ||
Note: These parameters are intended to be compatible with pre-made 250-mL amounts of [[Griffitts:Common media#LB Broth (1 L)|LB]] agar, such that usually 1 mL of stock solution is diluted into the [[Griffitts:Common media#LB Broth (1 L)|LB]] agar. For liquid [[Griffitts:Common media#LB Broth (1 L)|LB]] (typically 75 mL amounts) 0.3 mL of the stock solution will usually be added. | Note: These parameters are intended to be compatible with pre-made 250-mL amounts of [[Griffitts:Common media#LB Broth (1 L)|LB]] agar, such that usually 1 mL of stock solution is diluted into the [[Griffitts:Common media#LB Broth (1 L)|LB]] agar. For liquid [[Griffitts:Common media#LB Broth (1 L)|LB]] (typically 75 mL amounts) 0.3 mL of the stock solution will usually be added. Exceptions: Gentamycin, Nalidixic Acid, and Rifampicin. | ||
==DNA== | ==DNA== | ||
===10 mM dNTPs=== | ===10 mM dNTPs=== | ||
* Thaw and mix (stir) 100 mM stock solutions of dGTP, dATP, dTTP, dCTP (NEB; in freezer) | * Thaw and mix (stir) 100 mM stock solutions of dGTP, dATP, dTTP, dCTP (NEB; in freezer) | ||
* | * Master Mix: | ||
** 60 μl | ** 60 μl sterile dH<sub>2</sub>O | ||
** 10 μl [[Wikipedia:Guanine|dGTP]] | ** 10 μl [[Wikipedia:Guanine|dGTP]] | ||
** 10 μl [[Wikipedia:Adenine|dATP]] | ** 10 μl [[Wikipedia:Adenine|dATP]] | ||
** 10 μl [[Wikipedia:Thymine|dTTP]] | ** 10 μl [[Wikipedia:Thymine|dTTP]] | ||
** 10 μl [[Wikipedia:Cytosine|dCTP]] | ** 10 μl [[Wikipedia:Cytosine|dCTP]] | ||
Note: Store in the | * Dispense 100-μL aliquots into 1.7-mL tubes | ||
Note: Store in the –20°C freezer in the box marked "PCR Supplies" | |||
===10X ''Taq'' buffer=== | ===10X ''Taq'' buffer=== | ||
* Label tubes: 10X ''Taq'' buffer | * Label tubes: 10X ''Taq'' buffer | ||
* Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer) | * Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer) | ||
* Aliquot 375 μL of "Thermo pol buffer" into tubes—do '''not''' dilute! | * Aliquot 375 μL of "Thermo pol buffer" into tubes—do '''not''' dilute! | ||
Note: Store in the | Note: Store in the –20°C freezer in the box marked "PCR Supplies" | ||
===10X ''Pfx'' buffer=== | ===10X ''Pfx'' buffer=== | ||
* Label tubes: 10X ''Pfx'' buffer | * Label tubes: 10X ''Pfx'' buffer | ||
* Thaw and mix (stir) "''Pfx50'' 10X PCR Buffer" (NEB; in freezer) | * Thaw and mix (stir) "''Pfx50'' 10X PCR Buffer" (NEB; in freezer) | ||
* Aliquot 375 μL of "''Pfx50'' 10X PCR Buffer" into tubes—do '''not''' dilute! | * Aliquot 375 μL of "''Pfx50'' 10X PCR Buffer" into tubes—do '''not''' dilute! | ||
Note: Store in the | Note: Store in the –20°C freezer in the box marked "PCR Supplies" | ||
===5X ''Q5'' buffer=== | |||
* Label tubes: 5X ''Q5'' buffer | |||
* Thaw and mix (stir) "''Q5'' 5X PCR Buffer" (NEB; in freezer) | |||
* Aliquot 100 μL of "''Q5'' 5X PCR Buffer" into tubes—do '''not''' dilute! | |||
Note: Store in the –20°C freezer in the box marked "PCR Supplies" | |||
===8X DNA loading=== | ===8X DNA loading=== | ||
* 40% [[Griffitts:Stock solutions#80% Glycerol (75 mL)|glycerol]] | * 40% [[Griffitts:Stock solutions#80% Glycerol (75 mL)|glycerol]] | ||
* 1/50 dilution of [[Griffitts:Common buffers#2% bromophenol blue|2% bromophenol blue]] | * 1/50 dilution of [[Griffitts:Common buffers#2% bromophenol blue|2% bromophenol blue]] | ||
===DNA ladder=== | ===DNA ladder=== | ||
* | * 140 μL ddH<sub>2</sub>0 | ||
* | * 40 μL [[Griffitts:Common buffers#8X DNA loading|8X DNA loading]] buffer | ||
* 20 μL 1 kb ladder from NEB | * 20 μL 1 kb ladder from NEB | ||
* 2 μL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|0.25 M EDTA]]<br> | * 2 μL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|0.25 M EDTA]]<br> | ||
Note: use | Note: use 4 μL per lane; 3kb band = 50 ng | ||
===Ethidium Bromide (1 mL)=== | |||
* 100 μL 10 mg/mL ethidium bromide | |||
** This is stored on the middle shelf of the refrigerator on the right | |||
* 900 μL ddH<sub>2</sub>O | |||
Note: use 8 μL per 50 mL gel<br> | |||
WARNING: Always wear gloves when handling ethidium bromide | |||
===Ethidium Bromide (10 mL)=== | ===Ethidium Bromide (10 mL)=== | ||
* 10 mL ddH<sub>2</sub> | * 10 mL ddH<sub>2</sub>O | ||
* 10 mg ethidium bromide<br> | * 10 mg ethidium bromide<br> | ||
Note: use 8 μL per 50 mL gel | Note: use 8 μL per 50 mL gel<br> | ||
WARNING: Always wear gloves when handling ethidium bromide | |||
==Media Additives== | ==Media Additives== | ||
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===1 M Sodium Citrate (40 mL)=== | ===1 M Sodium Citrate (40 mL)=== | ||
* | * 35 mL ddH<sub>2</sub>O | ||
* 11.76 g sodium citrate tribasic dihydrate (Na<sub>3</sub>C<sub>6</sub>H<sub>5</sub>O<sub>7</sub>·2H<sub>2</sub>O) | * 11.76 g sodium citrate tribasic dihydrate (Na<sub>3</sub>C<sub>6</sub>H<sub>5</sub>O<sub>7</sub>·2H<sub>2</sub>O) | ||
* pH to 5.7 with HCl | * pH to 5.7 with HCl (~1 mL) | ||
* QS to 40 mL with ddH<sub>2</sub>O | |||
Sterile filter | Sterile filter | ||
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* 10 mL N,N-dimethylformamide (DMF)(in fume hood) | * 10 mL N,N-dimethylformamide (DMF)(in fume hood) | ||
* 0.2 g 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal)(in freezer) | * 0.2 g 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal)(in freezer) | ||
Store at | Store at –20°C | ||
===0.2 M IPTG=== | ===0.2 M IPTG=== | ||
* 5 mL ddH<sub>2</sub>O | * 5 mL ddH<sub>2</sub>O | ||
* 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer) | * 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer) | ||
Sterile filter, aliquot, and store at | Sterile filter, aliquot, and store at –20°C | ||
===1 M Copper (II) sulfate (50 mL)=== | ===1 M Copper (II) sulfate (50 mL)=== | ||
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* 771 mg Dithiothreitol (DTT; in fridge) | * 771 mg Dithiothreitol (DTT; in fridge) | ||
Aliquot 500 μL into Eppendorf tubes | Aliquot 500 μL into Eppendorf tubes | ||
Store in | Store in –20°C | ||
===Orcinol spray=== | ===Orcinol spray=== |
Latest revision as of 14:26, 13 December 2013
Antibiotics
Ampicillin (40 mL)
For a stock solution of 25 mg/mL:
- 1 g Ampicillin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Chloramphenicol (40 mL)
For a stock solution of 7.5 mg/mL:
- 300 mg Chloramphenicol
- 40 mL 50% Ethanol
NOTE: Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at 4°C
Erythromycin (20 mL)
For a stock solution of 10 mg/mL:
- 200 mg Erythromycin
- 20 mL 100% ethanol (in fume hood)
- Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at –20°C
Gentamicin (20 mL)
For a stock solution of 7.5 mg/mL:
- 150 mg Gentamicin
- 20 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Hygromycin (40 mL)
For a stock solution of 5 mg/mL:
- 200 mg Hygromycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Kanamycin (40 mL)
For a stock solution of 7.5 mg/mL:
- 300 mg Kanamycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Nalidixic acid (30 mL)
For a stock solution of 30 mg/mL:
- 900 mg Nalidixic Acid
- 360 mg NaOH
- 30 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Neomycin (40 mL)
For a stock solution of 25 mg/mL:
- 1 g Neomycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Phosphomycin
For a stock solution of 25 mg/mL:
- 1 g Phosphomycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Rifampicin (20 mL)
For a stock solution of 50 mg/mL:
- 1 g Rifampicin
- 20 mL N,N-dimethylformamide (DMF; in fume hood)
NOTE: Do not filter sterilize—the DMF will dissolve the filter
NOTE: Store at –20°C
Spectinomycin (40 mL)
For a stock solution of 20 mg/mL:
- 800 mg Spectinomycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Streptomycin (40 mL)
For a stock solution of 50 mg/mL:
- 2 g Streptomycin
- 40 mL ddH2O
- Filter sterilize
NOTE: Store at 4°C
Tetracycline (40 mL)
For a stock solution of 2.5 mg/mL:
- 100 mg Tetracycline
- 40 mL 50% Ethanol
NOTE: Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at 4°C
Triclosan (40 mL)
For a stock solution of 10 mg/mL:
NOTE: Do not filter sterilize—the ethanol may dissolve the filter
NOTE: Store at 4°C
Trimethoprim (40 mL)
For a stock solution of 6.25 mg/mL:
- 250 mg Trimethoprim
- 40 mL 100% DMSO
NOTE: Do not filter sterilize—the DMSO may dissolve the filter
NOTE: Store at 4°C
Antibiotics Table
Antibiotic | Abbr. | Stripe | Stock | E. coli | S. meliloti | Solvent | Storage |
---|---|---|---|---|---|---|---|
Ampicillin | Ap | BLACK | 25 mg/mL | 100 μg/mL (250X) | N/A | ddH2O | 4°C |
Chloramphenicol | Cm | BROWN | 7.5 mg/mL | 30 μg/mL (250X) | N/A | 50% EtOH | 4°C |
Erythromycin | Er | 10 mg/mL | N/A | N/A | 100% EtOH | –20°C | |
Gentamycin | Gm | PURPLE | 7.5 mg/mL | 3 μg/mL (2500X) | 15 μg/mL (500X) | ddH2O | 4°C |
Hygromycin | Hy | 5 mg/mL | 20 μg/mL (250X) | 20 μg/mL (250X) | ddH2O | 4°C | |
Kanamycin | Km | YELLOW | 7.5 mg/mL | 30 μg/mL (250X) | Use Neomycin | ddH2O | 4°C |
Nalidixic acid | Na | 30 mg/mL | 30 μg/mL (1000X) | N/A | ddH2O | 4°C | |
Neomycin | Nm | GREEN | 25 mg/mL | Use Kanamycin | 100 μg/mL (250X) | ddH2O | 4°C |
Phosphomycin | Pm | 25 mg/mL | N/A | 100 μg/mL (250X) | ddH2O | 4°C | |
Rifampicin | Rf | 50 mg/mL | N/A | 100 μg/mL (500X) | DMF | –20°C | |
Spectinomycin | Sp | ORANGE | 20 mg/mL | 80 μg/mL (250X) | 80 μg/mL (250X) | ddH2O | 4°C |
Streptomycin | Sm | BLUE | 50 mg/mL | N/A | 200 μg/mL (250X) | ddH2O | 4°C |
Tetracycline | Tc | RED | 2.5 mg/mL | 5 μg/mL (500X) | 5 μg/mL (500X) | 50% EtOH | 4°C |
Triclosan | Tr | 10 mg/mL | N/A | N/A | 100% EtOH | 4°C | |
Trimethoprim | Tm | 6.25 mg/mL | N/A | 12.5 μg/mL (500X) |
100% DMSO | 4°C |
Note: These parameters are intended to be compatible with pre-made 250-mL amounts of LB agar, such that usually 1 mL of stock solution is diluted into the LB agar. For liquid LB (typically 75 mL amounts) 0.3 mL of the stock solution will usually be added. Exceptions: Gentamycin, Nalidixic Acid, and Rifampicin.
DNA
10 mM dNTPs
- Thaw and mix (stir) 100 mM stock solutions of dGTP, dATP, dTTP, dCTP (NEB; in freezer)
- Master Mix:
- Dispense 100-μL aliquots into 1.7-mL tubes
Note: Store in the –20°C freezer in the box marked "PCR Supplies"
10X Taq buffer
- Label tubes: 10X Taq buffer
- Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer)
- Aliquot 375 μL of "Thermo pol buffer" into tubes—do not dilute!
Note: Store in the –20°C freezer in the box marked "PCR Supplies"
10X Pfx buffer
- Label tubes: 10X Pfx buffer
- Thaw and mix (stir) "Pfx50 10X PCR Buffer" (NEB; in freezer)
- Aliquot 375 μL of "Pfx50 10X PCR Buffer" into tubes—do not dilute!
Note: Store in the –20°C freezer in the box marked "PCR Supplies"
5X Q5 buffer
- Label tubes: 5X Q5 buffer
- Thaw and mix (stir) "Q5 5X PCR Buffer" (NEB; in freezer)
- Aliquot 100 μL of "Q5 5X PCR Buffer" into tubes—do not dilute!
Note: Store in the –20°C freezer in the box marked "PCR Supplies"
8X DNA loading
- 40% glycerol
- 1/50 dilution of 2% bromophenol blue
DNA ladder
- 140 μL ddH20
- 40 μL 8X DNA loading buffer
- 20 μL 1 kb ladder from NEB
- 2 μL 0.25 M EDTA
Note: use 4 μL per lane; 3kb band = 50 ng
Ethidium Bromide (1 mL)
- 100 μL 10 mg/mL ethidium bromide
- This is stored on the middle shelf of the refrigerator on the right
- 900 μL ddH2O
Note: use 8 μL per 50 mL gel
WARNING: Always wear gloves when handling ethidium bromide
Ethidium Bromide (10 mL)
- 10 mL ddH2O
- 10 mg ethidium bromide
Note: use 8 μL per 50 mL gel
WARNING: Always wear gloves when handling ethidium bromide
Media Additives
1 M Calcium Chloride (75 mL)
- 75 mL ddH2O
- 11.027 g calcium chloride dihydrate (CaCl2·2H2O)
Autoclave
2 M Magnesium Chloride (75 mL)
- 75 mL ddH2O
- 30.4965 g magnesium chloride hexahydrate (MgCl2·6H2O)
Autoclave
1 M Sodium Citrate (40 mL)
- 35 mL ddH2O
- 11.76 g sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O)
- pH to 5.7 with HCl (~1 mL)
- QS to 40 mL with ddH2O
Sterile filter
2% X-gal
- 10 mL N,N-dimethylformamide (DMF)(in fume hood)
- 0.2 g 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal)(in freezer)
Store at –20°C
0.2 M IPTG
- 5 mL ddH2O
- 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer)
Sterile filter, aliquot, and store at –20°C
1 M Copper (II) sulfate (50 mL)
- 50 mL ddH2O
- 7.98 g CuSO4
Sterile filter
10% SDS (75 mL)
- 75 mL ddH2O
- 7.5 g Sodium dodecyl sulfate (SDS)
Do not sterilize
20% SDS (75 mL)
- 75 mL ddH2O
- 15 g Sodium dodecyl sulfate (SDS)
Do not sterilize
30% Acrylamide (75 mL)
- 75 mL ddH2O
- 22.5 g acrylamide
40% Acrylamide (75 mL)
- 75 mL ddH2O
- 30 g acrylamide
10% Ammonium persulfate (75 mL)
- 75 mL ddH2O
- 7.5 g ammonium persulfate (APS)
5 M NaCl (75 mL)
- 75 mL ddH2O
- 21.92 g NaCl
Autoclave
80% Glycerol (75 mL)
- 15 mL ddH2O
- 60 mL glycerol
Microwave for 30-second intervals on high until dissolved
Autoclave
1 M DTT (5 mL)
- 5 mL ddH2O
- 771 mg Dithiothreitol (DTT; in fridge)
Aliquot 500 μL into Eppendorf tubes Store in –20°C
Orcinol spray
- 5 mL ddH2O
- 25 mL methanol (in fume hood)
- 10 mL sulfuric acid (H2SO4 (in fume hood—be careful!)
- 200 mg orcinol