(r69) ProtocolsAntibioticStockSolutions < Lab

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---+ Media amendments 

---++ Antibiotic concentrations and stock solutions
This table lists “standard” concentrations of antibiotics commonly used in microbiology laboratories. These concentrations apply to _E. coli_. Antibiotic selection in other organisms may use different concentrations, so check primary literature for more info.  
%TABLE{headeralign="left,center,center,center,center,left" dataalign="left,center,center,center,center,left,center,left"}%
| *Reagent* | *Abb* | *Stock Conc.* | *Working Conc.* | *Dilution* | *Solvent* | *MSDS* | *Notes* |
| Ampicillin | Amp | 100 mg/mL | 100 µg/mL | 1,000× | ddH<sub>2</sub>O | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=A9518&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fa9518%3Flang%3Den][ link]] | Ampicillin degrades quickly in both plates and stock solutions. Culture plates with Amp can be stored at 4°C for about 2 weeks. Stock solutions can be stored at 4°C for 2 weeks but can last as long as 4-6 months when stored at -20°C. In general, use carbenicillin for ampicillin selection, because it is more chemically stable. |
| Carbenicillin | Crb | 100 mg/mL | 100 µg/mL | 1,000× | 50% EtOH | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=C1389&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fc1389%3Flang%3Den][link]] | Carbenicillin can be used in place of Ampicillin at the same working concentration. Carbenicillin is more stable than Ampicillin, and is generally preferable. |
| Chloramphenicol | Cam | 20 mg/mL | 20 µg/mL | 1,000× | EtOH | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=C0378&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fc0378%3Flang%3Den][link]] | |
| Cycloheximide | Chx | 100 mg/mL | 100 µg/mL | 1,000× | 100% DMSO | [[http://www.sciencelab.com/msds.php?msdsId=9923627][link]] | Anti-fungal, toxic to humans, use PPE! |
| D-Cycloserine | Dcs | 4 mg/mL | 4 µg/mL | 1,000× | ddH<sub>2</sub>O | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=30020&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2F30020%3Flang%3Den][link]] | Degrades noticeably over the course of 1 week at 4°C. D-cycloserine is best used within two days of pouring. A single small-scale experiment found 6-10x as many _E. coli_ colonies on 8-day old MG-Dcs plates that had spent 3 of those days at -20°C and 5 at 4°C, compared to what was found on 1 day old MG-Dcs. |
| Erythromycin  | Ery | 250 mg/mL | 250 µg/mL | 1,000x | EtOH | [[http://www.sigmaaldrich.com/catalog/product/sigma/e5389?lang=en®ion=US][link]] | May crystallize when added to liquid culture. Still works. Selection strength may vary in non-model bacteria  |
| Gentamicin | Gen | 20 mg/mL | 20 µg/mL | 1,000× | ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927175][link]] | For non-canonical experiments: <br />Use 20 µg/mL in culture from 20mg/mL 1000× stock <br />E. coli may spontaneously develop resistance on the chromosome(?) |
| G418 | G418 | 200 mg/mL | 200 µg/mL | 1000x | ddH<sub>2</sub>O | [[https://www.goldbio.com/product/3022/g418-sulfate][link]] | Geneticin generic. Powder store RT. Stock solution at -20°C |
| Kanamycin | Kan | 50 mg/mL | 50 µg/mL | 1,000× |  ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927199][link]] | Plates start to lose sensitivity after 2 weeks; [[https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/k1377pis.pdf][recommended]] Kanamycin plates only last a few weeks at 4°C; best to make fresh Kan plates every 2 - 4 weeks. |
| Nalidixic Acid | Nal | 30 mg/mL | 30 µg/mL | 1,000× |  1M NaOH | [[http://www.sciencelab.com/msds.php?msdsId=9926169][link]] | |
| Novobiocin | Nov | 200 mg/mL | 200 µg/mL | 1,000× | ddH<sub>2</sub>O | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=N1628&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fn1628%3Flang%3Den][link]] | |
| Rifampicin | Rif | 50 mg/mL | 100 µg/mL | 500× | MeOH + drops of 10M NaOH |  [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=R3501&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fr3501%3Flang%3Den][link]] |  <b> While dissolving Rifampicin in MeOH, there might be a small residue left at the bottom which may not go into solution even after adding a few drops of NaOH. </b> Toxic to humans, use PPE! Rifampicin is TOXIC to humans, light-sensitive, and degrades in solution rather quickly. It needs to be stored wrapped in foil and replaced every 2 months. |
| Spectinomycin | Spec | 60 mg/mL | 60 µg/mL | 1,000× | ddH<sub>2</sub>O | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=S4014&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fs4014%3Flang%3Den][link]] | Spontaneous mutations in the chromosome can yield resistant colonies. *Recommend using at a 120 µg/mL working concentration when cloning to decrease this background!! (double what you would normally add to your media)*  | 
| Streptomycin | Str | 100 mg/mL | 100 µg/mL | 1,000× | ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927414][link]] | |
| Tetracycline | Tet | 10 mg/mL | 10 µg/mL | 1,000× |  70% EtOH | [[http://www.sciencelab.com/msds.php?msdsId=9925200][link]] | Light sensitive, stability &lt; 2 weeks |
| Trimethoprim | TMP |  10 mg/mL | variable? | variable | DMSO | [[https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/t7883pis.pdf][link]] | [[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1072810/][Both 10 and 20 µg/ml listed for use. Use as counter selectable marker for mutations in _thyA_ requires presence of thymine/thymidine in media]] |
| Zeocin | Zeo | 25 mg/mL | 25 µg/mL | 1,000× | ddH<sub>2</sub>O | [[http://www.invivogen.com/MSDS/Zeocin_solution_SDS.pdf][link]]  | [[https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/transfection-protocol/zeocin.html][Product literature]] lists active range of 25 - 50 µg/ml for _E. coli_ and 50 - 300 in yeast. In our hands we note: <br />10 µg/mL is specifically used for growth of amberless strain A37. For yeast, 100µg / ml is sufficient. |

---++Other common media amendments

%TABLE{headeralign="left,center,center,center,center,left" dataalign="left,center,center,center,center,left,center,left"}%
| *Reagent* | *Abb* | *Stock Conc.* | *Working Conc.* | *Dilution* | *Solvent* | *MSDS* | *Notes* |
| 5-fluorouracil | 5FU | 13 mg/mL | 13 µg/mL | 1,000× | DMSO | [[https://www.fishersci.com/shop/products/5-fluorouracil-99-acros-organics-4/AC228440050][link]] | [[https://link.springer.com/article/10.1007%2Fs10529-014-1718-8][100mM stock concentration 100µM working concentration]] |
| Anhydrotetracycline | ATC | 0.1 mg/mL | 100 ng/mL | 1,000× | DMSO | [[https://www.caymanchem.com/pdfs/10009542.pdf][link]] | Can also make a 1mg/mL stock and dilute 1:10 for a final concentration of 0.1mg/mL |
| 3&prime;-Azido-2&prime;,3&prime;-dideoxythymidine | AZT | 10 mg/mL | 200 µg/mL | 50× | ddH<sub>2</sub>O | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=11546&brand=FLUKA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Ffluka%2F11546%3Flang%3Den][link]] | Can substitute pharma grade Zidovudine. Soluble up to 20 mg/mL in ddH<sub>2</sub>O. Very unstable in solution. Store at –20°C and activity will still decrease over weeks of storage.  |
| Bromo-chloro-indolyl-galactopyranoside | X-gal | 40 mg/mL | 80 µg/mL | 500× | Dimethylformamide (DMF) | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=B4252&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fb4252%3Flang%3Den][link]] | X-gal is a histochemical substrate for &beta;-galactosidase, which cleaves X-Gal to yield an insoluble blue precipitate. |
| Diaminopimelic acid | DAP | 60 mM | 0.3 mM | 200× | ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927175][link]] | Use 5 µl stock per 1 ml of media. Dissolve ~30min shaking at 37C |
| Isopropyl &beta;-D-1-thiogalactopyranoside  | IPTG | 0.1 M | 0.1 mM | 1,000× | ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9924418][link]] | Light sensitive, MW: 238.31 g/mol |
| Triphenyl Tetrazolium Chloride | TTC | 5% (50 mg/mL) |  0.05% (50 µg/mL) | 1,000× |  ddH<sub>2</sub>O | [[https://www.caymanchem.com/msdss/17342m.pdf][link]] | Used in growth/sugar-fermentation indicator agars. Filter sterilize. Light sensitive. Store stock solution at 4°C wrapped in foil. Use at ten-fold higher concentration in motility agar (0.5%). |


Working concentrations are for liquid or solid media.

---++ Preparation

  * Generally, prepare 30-50 mL of solution in a 50 mL conical tube. Then, filter sterilize solutions by pushing them through a 50 mL syringe fitted with a 0.22 µm filter made of a material appropriate for the solvent. Compounds dissolved in Ethanol or DMSO do not require filter sterilization. Aliquot into 1.5 mL portions in 1.7 mL tubes using the repeat pipettor. Label with a permanent marker (one that won't wash off with ethanol). Store at -20°C in the dark.

---++Non-canonical amino acid stock solutions
|*Amino Acid<br>(Abbreviation)*|*CAS Number*|*Solvent*|*MW<br>(g/mol)*|*[Stock]*|*[Stock]<br>(mg/mL)*|*[Culture]*|*Notes*|
|!Y|60-18-4|Water|181.19|10mM|1.812|250µM|Requires heating to 70ºC to dissolve<br> Stock precipitates out of solution at or below room temperature|
|!IodoY|70-78-0|Water|307.09|10mM|3.071|250µM|Dissolves at room temperature with ~1 hour of shaking|
|!AminoY <small>(FAA)</small>|300-34-5||196.203|||||
|!AminoY <small>(HS)</small>|23279-22-3|Water|287.14|10mM||250µM|Dissolves readily without shaking or heat|
|!BromoY|38739-13-8|Water|260.0846|10mM||250µM||
|!ChloroY|7423-93-0|Water|215.63|10mM||250µM||
|!NitroY|621-44-3|Water|226.2|10mM|2.262|250µM|Will dissolve with heating to 50ºC, no shaking|
|!ONBY <small>(FAA)</small>|184591-51-3||316.31||||Light sensitive|
|!ONBY <small>(S)</small>|207727-86-4|50% DMSO<br>50% Water<br>(exothermic)|352.77|50 mM||250µM|Light sensitive<br>Requires adding !OH<sup>-</sup> to dissolve|
|!AzF|33173-53-4|10% DMSO<br>90% Water|206.204|10mM|2.062|250µM|Light sensitive<br>Requires heating and shaking to dissolve|
|!CNF|167479-78-9|Water|190.2|10mM|1.902|250µM|Dissolves at room temperature with ~15 minutes of shaking|
|<small>L</small>-DOPA|59-92-7|Water|197.1879|10mM|1.972|250µM|Oxidizes quickly and turns black in solution|
|!5HTP|4350-09-8|Water|220.2246|10mM|2.202|250µM||
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<b>FAA</b>: free amino acid<br>
<b>S</b>: salt<br>
<b>HS</b>: hydrated salt<br>
Attachments
Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
docx	Making_stock_antibiotics_and_other_reagents.docx	r1	manage	89.7 K	2019-01-11 - 21:56	KateElston	Easy spreadsheet that tells you exactly how to make the most common stock chemicals
Barrick Lab > ProtocolList > ProtocolsGeneGorgingMarker > ProtocolsAntibioticStockSolutions
Topic revision: r69 - 2024-09-20 - 20:17:16 - Main.AlexaMorton