Griffitts:Stock solutions: Difference between revisions
(48 intermediate revisions by 3 users not shown) | |||
Line 1: | Line 1: | ||
{{Griffitts}} | <div class="noprint"> | ||
{{Template:Griffitts}} | |||
</div> | |||
==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: | ||
* 40 mL [[Wikipedia:Ethanol|Ethanol]] | * 300 mg [[Wikipedia:Chloramphenicol|Chloramphenicol]] | ||
** Do '''not''' filter sterilize | * 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)=== | ||
For a stock solution of 10 mg/mL: | |||
* 200 mg [[Wikipedia:Erythromycin|Erythromycin]] | |||
* 20 mL 100% ethanol (in fume hood) | |||
* Do '''not''' filter sterilize—the ethanol may dissolve the filter | |||
NOTE: Store at –20°C | |||
===Gentamicin (40 mL)=== | |||
For a stock solution of 7.5 mg/mL: | |||
* 300 mg [[Wikipedia:Gentamicin|Gentamicin]] | |||
* 40 mL ddH<sub>2</sub>O | * 40 mL ddH<sub>2</sub>O | ||
* Filter sterilize | * Filter sterilize | ||
NOTE: Store at 4°C | |||
===Hygromycin (40 mL)=== | ===Hygromycin (40 mL)=== | ||
For a stock solution of 5 mg/mL: | |||
* 200 mg [[Wikipedia:Hygromycin|Hygromycin]] | |||
* 40 mL ddH<sub>2</sub>O | |||
* 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 | ||
** Store | NOTE: Store at 4°C | ||
===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 | |||
* 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 in | 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)=== | |||
For a stock solution of 50 mg/mL: | |||
* 1 g [[Wikipedia:Rifampicin|Rifampicin]] | |||
* 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: | ||
* 40 mL [[Wikipedia:Ethanol|Ethanol]] | * 100 mg [[Wikipedia:Tetracycline|Tetracycline]] | ||
** Do '''not''' filter sterilize | * 40 mL 50% [[Wikipedia:Ethanol|Ethanol]] | ||
** Store | NOTE: Do '''not''' filter sterilize—the ethanol may dissolve the filter<br> | ||
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== | |||
{| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable" | |||
|+'''Antibiotic Specifications''' | |||
|- | |||
! width="120" style="background:#efefef;" | Antibiotic | |||
! width="30" style="background:#efefef;" | Abbr. | |||
! width="90" style="background:#efefef;" | Stripe | |||
! width="90" style="background:#efefef;" | Stock | |||
! width="120" style="background:#efefef;" | ''E. coli'' | |||
! width="120" style="background:#efefef;" | ''S. meliloti'' | |||
! width="90" style="background:#efefef;" | Solvent | |||
! width="90" style="background:#efefef;" | Storage | |||
|- | |||
| Ampicillin | |||
| Ap | |||
! style="background:#000000;" | <font color=white>'''BLACK'''</font> | |||
| 25 mg/mL | |||
| 100 μg/mL (250X) | |||
| N/A | |||
| ddH<sub>2</sub>O | |||
| 4°C | |||
|- | |||
| Chloramphenicol | |||
| Cm | |||
! style="background:#8B4513;" | <font color=white>'''BROWN'''</font> | |||
| 7.5 mg/mL | |||
| 30 μg/mL (250X) | |||
| N/A | |||
| 50% EtOH | |||
| 4°C | |||
|- | |||
| Erythromycin | |||
| Er | |||
| <center>(none)</center> | |||
| 10 mg/mL | |||
| N/A | |||
| N/A | |||
| 100% EtOH | |||
| –20°C | |||
|- | |||
| Gentamycin | |||
| Gm | |||
! style="background:#9400D3;" | <font color=white>'''PURPLE'''</font> | |||
| 7.5 mg/mL | |||
| 3 μg/mL (2500X) | |||
| 15 μg/mL (500X) | |||
| ddH<sub>2</sub>O | |||
| 4°C | |||
|- | |||
| Hygromycin | |||
| Hy | |||
| <center>(none)</center> | |||
| 5 mg/mL | |||
| 20 μg/mL (250X) | |||
| 20 μg/mL (250X) | |||
| ddH<sub>2</sub>O | |||
| 4°C | |||
|- | |||
| Kanamycin | |||
| Km | |||
! style="background:#FFD700;" | <font color=white>'''YELLOW'''</font> | |||
| 7.5 mg/mL | |||
| 30 μg/mL (250X) | |||
| '''Use Neomycin''' | |||
| ddH<sub>2</sub>O | |||
| 4°C | |||
|- | |||
| Nalidixic acid | |||
| Na | |||
| <center>(none)</center> | |||
| 30 mg/mL | |||
| 30 μg/mL (1000X) | |||
| N/A | |||
| ddH<sub>2</sub>O | |||
| 4°C | |||
|- | |||
| Neomycin | |||
| Nm | |||
! 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 | |||
| 100 μg/mL (250X) | |||
| ddH<sub>2</sub>O | |||
| 4°C | |||
|- | |||
| Rifampicin | |||
| Rf | |||
| <center>(none)</center> | |||
| 50 mg/mL | |||
| N/A | |||
| 100 μg/mL (500X) | |||
| DMF | |||
| –20°C | |||
|- | |||
| Spectinomycin | |||
| Sp | |||
! style="background:#FF8C00;" | <font color=white>'''ORANGE'''</font> | |||
| 20 mg/mL | |||
| 80 μg/mL (250X) | |||
| 80 μg/mL (250X) | |||
| ddH<sub>2</sub>O | |||
| 4°C | |||
|- | |||
| Streptomycin | |||
| Sm | |||
! style="background:#0000FF;" | <font color=white>'''BLUE'''</font> | |||
| 50 mg/mL | |||
| N/A | |||
| 200 μg/mL (250X) | |||
| ddH<sub>2</sub>O | |||
| 4°C | |||
|- | |||
| Tetracycline | |||
| Tc | |||
! style="background:#FF0000;" | <font color=white>'''RED'''</font> | |||
| 2.5 mg/mL | |||
| 5 μg/mL (500X) | |||
| 5 μg/mL (500X) | |||
| 50% EtOH | |||
| 4°C | |||
|- | |||
| Triclosan | |||
| Tr | |||
| <center>(none)</center> | |||
| 10 mg/mL | |||
| N/A | |||
| N/A | |||
| 100% EtOH | |||
| 4°C | |||
|- | |||
| Trimethoprim | |||
| Tm | |||
| <center>(none)</center> | |||
| 6.25 mg/mL | |||
| N/A | |||
| 12.5 μg/mL (500X)<br><center>or</center>25 μg/mL (250X) | |||
| 100% DMSO | |||
| 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. Exceptions: Gentamycin, Nalidixic Acid, and Rifampicin. | |||
==DNA== | ==DNA== | ||
===10 mM dNTPs=== | ===10 mM dNTPs=== | ||
* Thaw and mix 100 mM stock solutions of | * 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| | ** 10 μl [[Wikipedia:Guanine|dGTP]] | ||
** 10 μl [[Wikipedia:Adenine| | ** 10 μl [[Wikipedia:Adenine|dATP]] | ||
** 10 μl [[Wikipedia:Thymine| | ** 10 μl [[Wikipedia:Thymine|dTTP]] | ||
** 10 μl [[Wikipedia:Cytosine| | ** 10 μl [[Wikipedia:Cytosine|dCTP]] | ||
* Dispense 100-μL aliquots into 1.7-mL tubes | |||
Note: Store in the –20°C freezer in the box marked "Other Enzymes" | |||
===10X ''Taq'' buffer=== | ===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 "Other Enzymes" | |||
===10X ''Pfx'' buffer=== | ===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 "Other Enzymes" | |||
===8X DNA loading=== | |||
* 40% [[Griffitts:Stock solutions#80% Glycerol (75 mL)|glycerol]] | |||
* 1/50 dilution of [[Griffitts:Common buffers#2% bromophenol blue|2% bromophenol blue]] | |||
===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 | |||
* 2 μL [[Griffitts:Common buffers#0.25 M EDTA (75 mL)|0.25 M EDTA]]<br> | |||
Note: use 5 μL per lane; 3kb band = 60 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)=== | |||
* 10 mL ddH<sub>2</sub>O | |||
* 10 mg ethidium bromide<br> | |||
Note: use 8 μL per 50 mL gel<br> | |||
WARNING: Always wear gloves when handling ethidium bromide | |||
==Media Additives== | ==Media Additives== | ||
===1 M Calcium Chloride (75 mL)=== | ===1 M Calcium Chloride (75 mL)=== | ||
* 75 mL | * 75 mL ddH<sub>2</sub>O | ||
* 11.027 g CaCl<sub>2</sub>·2H<sub>2</sub>O | * 11.027 g calcium chloride dihydrate (CaCl<sub>2</sub>·2H<sub>2</sub>O) | ||
Autoclave | |||
===2 M Magnesium Chloride (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 30.4965 g magnesium chloride hexahydrate (MgCl<sub>2</sub>·6H<sub>2</sub>O) | |||
Autoclave | Autoclave | ||
===X-gal=== | ===1 M Sodium Citrate (40 mL)=== | ||
===IPTG=== | * 35 mL ddH<sub>2</sub>O | ||
===Copper sulfate=== | * 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) | ||
===SDS=== | * pH to 5.7 with HCl (~1 mL) | ||
* QS to 40 mL with ddH<sub>2</sub>O | |||
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 ddH<sub>2</sub>O | |||
* 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 ddH<sub>2</sub>O | |||
* 7.98 g CuSO<sub>4</sub> | |||
Sterile filter | |||
===10% [[SDS]] (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 7.5 g Sodium dodecyl sulfate ([[SDS]]) | |||
Do '''not''' sterilize | |||
===20% [[SDS]] (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 15 g Sodium dodecyl sulfate ([[SDS]]) | |||
Do '''not''' sterilize | |||
===30% Acrylamide (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 22.5 g acrylamide | |||
===40% Acrylamide (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 30 g acrylamide | |||
===10% Ammonium persulfate (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 7.5 g ammonium persulfate (APS) | |||
===5 M NaCl (75 mL)=== | |||
* 75 mL ddH<sub>2</sub>O | |||
* 21.92 g NaCl | |||
Autoclave | |||
===80% Glycerol (75 mL)=== | |||
* 15 mL ddH<sub>2</sub>O | |||
* 60 mL glycerol | |||
Microwave for 30-second intervals on high until dissolved<br> | |||
Autoclave | |||
===1 M DTT (5 mL) === | |||
* 5 mL ddH<sub>2</sub>O | |||
* 771 mg Dithiothreitol (DTT; in fridge) | |||
Aliquot 500 μL into Eppendorf tubes | |||
Store in –20°C | |||
===Orcinol spray=== | |||
* 5 mL ddH<sub>2</sub>O | |||
* 25 mL methanol (in fume hood) | |||
* 10 mL sulfuric acid (H<sub>2</sub>SO<sub>4</sub> (in fume hood—be careful!) | |||
* 200 mg orcinol |
Revision as of 15:41, 29 February 2012
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 (40 mL)
For a stock solution of 7.5 mg/mL:
- 300 mg Gentamicin
- 40 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 "Other Enzymes"
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 "Other Enzymes"
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 "Other Enzymes"
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 5 μL per lane; 3kb band = 60 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