Barrick Lab :: ProtocolsAntibioticStockSolutions

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---+ Media Amendment Stock Solutions

%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]] |
| 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]] | |
| 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  |
| 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. |
| 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]] | |
| 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 |
| 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 |
| 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 |
| 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 |
| 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]] 4C storage time is ~1 month|
| 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]] |  Toxic to humans, use PPE! |
| 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]] | Common to see background growth of cells on selective plates. Best selection with twice the usual concentration of antibiotics (2×). | 
| 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 |
| 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%). |
| 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 | 10 mg/mL | 10 µg/mL | 1,000× | ddH<sub>2</sub>O | [[http://www.invivogen.com/MSDS/Zeocin_solution_SDS.pdf][link]]  | Some labs report using a higher concentration for selection. <br />10 µg/mL is specifically used for growth of amberless strain A37 |

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. 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.

---++ Other considerations

   * 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.
   * 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.
   * 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.
   * Kanamycin plates only last a few weeks at 4°C; best to make fresh Kan plates every 2 weeks.

---++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
This topic: Lab > WebHome > ProtocolList > ProtocolsGeneGorgingMarker > ProtocolsAntibioticStockSolutions
Topic revision: r56 - 2019-11-20 - DanielDeatherage