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| - | TAP medium
| + | <html> |
| | + | <script type="text/javascript" src="https://2011.igem.org/Team:Bilkent_UNAM_Turkey/js/dropdowncontent.js?action=raw&ctype=text/css\"></script> |
| | + | <link href='https://2011.igem.org/Team:Bilkent_UNAM_Turkey/css/tabbedContent.css?action=raw&ctype=text/css' rel='stylesheet' type='text/css'/> |
| | + | <script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.3/jquery.min.js'></script> |
| | + | <script type="text/javascript" src="https://2011.igem.org/Team:Bilkent_UNAM_Turkey/wordlist.js?action=raw&ctype=text/css\"></script> |
| | | | |
| - | from Gorman, D.S., and R.P. Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669.
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| | + | .left-menu noprint{ |
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| | + | } |
| | | | |
| - | This is probably the most widely-used medium at present for experimental work.
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| - | Make the following stock solutions:
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| | | | |
| - | 1. TAP salts
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| - |
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| - | NH4Cl 15.0 g
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| - | MgSO4 . 7H2O 4.0 g
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| - | CaCl2 . 2H2O 2.0 g
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| - | water to 1 liter
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| - |
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| - | 2. phosphate solution
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| - |
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| - | K2HPO4 28.8 g
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| - | KH2PO4 14.4 g
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| - | water to 100 ml
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| - |
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| - | 3. Hutner's trace elements
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| - |
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| - |
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| - |
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| - | To make the final medium, mix the following:
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| - |
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| - | 2.42 g Tris
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| - | 25 ml solution #1 (salts)
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| - | 0.375 ml solution #2 (phosphate)
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| - | 1.0 ml solution #3 (trace elements)
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| - | 1.0 ml glacial acetic acid
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| - | water to 1 liter
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| - |
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| - | For solid medium, add 15 g agar per liter
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| - |
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| - | Autoclave.
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| - |
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| - | For Tris-minimal medium omit the acetic acid and titrate the final solution to pH 7.0 with HCl
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| - |
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| - | Hutner's trace elements
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| - |
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| - | Hutner et al. (1950) Proc. Am. Philos. Soc. 94, 152-170
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| - |
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| - | This mixture is used both in TAP and in the Sueoka high salt medium.
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| - |
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| - | For a detailed analysis of how well this trace elements solution meets the nutritional requirements of C. reinhardtii, see Merchant et al. (2006) Biochim. Biophys. Acta 1763, 578-594.
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| - |
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| - |
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| - |
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| - | For 1 liter final mix, dissolve each compound in the volume of water indicated.
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| - |
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| - | The EDTA should be dissolved in boiling water, and the FeSO4 should be prepared last to avoid oxidation.
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| - | compound amount water
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| - | EDTA disodium salt 50 g 250 ml
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| - | ZnSO4 . 7 H2O 22 g 100 ml
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| - | H3BO3 11.4 g 200 ml
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| - | MnCl2 . 4 H2O 5.06 g 50 ml
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| - | CoCl2. 6 H2O 1.61 g 50 ml
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| - | CuSO4 . 5 H2O 1.57 g 50 ml
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| - | (NH4)6Mo7O24. 4 H2O 1.10 g 50 ml
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| - | FeSO4. 7 H2O 4.99 g 50 ml
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| - |
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| - | Mix all solutions except EDTA. Bring to boil, then add EDTA solution. The mixture should turn green. When everything is dissolved, cool to 70 degrees C. Keeping temperature at 70, add 85 ml hot 20% KOH solution (20 grams / 100 ml final volume). Do NOT use NaOH to adjust the pH.
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| - |
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| - | Bring the final solution to 1 liter total volume. It should be clear green initially. Stopper the flask with a cotton plug and let it stand for 1-2 weeks, shaking it once a day. The solution should eventually turn purple and leave a rust-brown precipitate, which can be removed by filtering through two layers of Whatman#1 filter paper, repeating the filtration if necessary until the solution is clear. Store refrigerated or frozen convenient aliquots. Some people shorten the time for formation of the precipiate by bubbling the solution with filtered air.
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| - |
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| - | If no precipitate forms, the solution is still usable. However, you might want to check the pH in this case and adjust it to around 7.0 using either KOH or HCl as needed.
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| - |
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| - | To prepare sulfur-free trace elements for hydrogen generation, the sulfate salts can be replaced with equimolar chloride salts (ZnCl2 10.0 g; CuCl2 . 2 H2O 1.00 g; FeCl2 . 4 H2O, 3.60 g). .
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| - | src="Transformation%20of%20Chlamydomonas%20with%20Glass%20Beads_dosyalar/image001.gif"
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| - | v:shapes="_x0000_i1025"><![endif]><span class=SpellE>Transformation</span> of
| |
| - | Chlamydomonas <span class=SpellE>with</span> <span class=SpellE>Glass</span> <span
| |
| - | class=SpellE>Beads</span><o:p></o:p></span></b></p>
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| | | | |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height: | + | <h1> Growth Conditions </h1> |
| - | normal'><span class=SpellE><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
| + | <b>Tris Acetate Phosphate (TAP) medium</b> |
| - | mso-fareast-font-family:"Times New Roman";mso-ansi-language:TR;mso-fareast-language:
| + | |
| - | TR'>contributed</span></span><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
| + | |
| - | mso-fareast-font-family:"Times New Roman";mso-ansi-language:TR;mso-fareast-language:
| + | |
| - | TR'> <span class=SpellE>by</span> Karen <span class=SpellE>Kindle</span><o:p></o:p></span></p>
| + | |
| | | | |
| - | <div class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
| |
| - | text-align:center;line-height:normal'><span style='font-size:12.0pt;font-family:
| |
| - | "Times New Roman","serif";mso-fareast-font-family:"Times New Roman";mso-ansi-language:
| |
| - | TR;mso-fareast-language:TR'>
| |
| | | | |
| - | <hr size=2 width="100%" align=center> | + | from Gorman, D.S., and R.P. Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669.<br> |
| | | | |
| - | </span></div> | + | This is probably the most widely-used medium at present for experimental work.<br> |
| | | | |
| - | <span style='font-size:12.0pt;font-family:"Times New Roman","serif";mso-fareast-font-family:
| + | Following solutions are prepared.<br> |
| - | "Times New Roman";mso-ansi-language:TR;mso-fareast-language:TR;mso-bidi-language:
| + | |
| - | AR-SA'>
| + | |
| | | | |
| - | <ul type=disc> | + | <br> 1. TAP salts |
| - | <li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;
| + | |
| - | line-height:normal;mso-list:l1 level1 lfo1;tab-stops:list 36.0pt'><span
| + | <br> NH4Cl 15.0 g |
| - | style='font-size:12.0pt;font-family:"Times New Roman","serif";mso-fareast-font-family:
| + | <br> MgSO4 . 7H2O 4.0 g |
| - | "Times New Roman";mso-ansi-language:TR;mso-fareast-language:TR'>1. <span
| + | CaCl2 . 2H2O 2.0 g |
| - | class=SpellE>Grow</span> <span class=SpellE>cells</span> in SGII+NH4NO3 <span | + | <br> water to 1 liter |
| - | class=SpellE>until</span> <span class=SpellE>they</span> <span
| + | <br> |
| - | class=SpellE>reach</span> a <span class=SpellE>density</span> of 1-2 x
| + | <br> 2. phosphate solution |
| - | 10^6/ml.<o:p></o:p></span></li>
| + | <br> |
| - | <li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto; | + | <br> K2HPO4 28.8 g |
| - | line-height:normal;mso-list:l1 level1 lfo1;tab-stops:list 36.0pt'><span
| + | <br> KH2PO4 14.4 g |
| - | style='font-size:12.0pt;font-family:"Times New Roman","serif";mso-fareast-font-family:
| + | <br> water to 100 ml |
| - | "Times New Roman";mso-ansi-language:TR;mso-fareast-language:TR'>2. <span
| + | <br> |
| - | class=SpellE>Spin</span> <span class=SpellE>down</span> <span
| + | <br>3. Hutner's trace elements |
| - | class=SpellE>cells</span> at <span class=SpellE>moderate</span> <span
| + | <br> |
| - | class=SpellE>speed</span> (5000 <span class=SpellE>rpm</span> <span
| + | <br> |
| - | class=SpellE>for</span> 5 <span class=SpellE>min</span> in a GSA <span
| + | |
| - | class=SpellE>type</span> rotor).<o:p></o:p></span></li>
| + | |
| - | <li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;
| + | |
| - | line-height:normal;mso-list:l1 level1 lfo1;tab-stops:list 36.0pt'><span
| + | |
| - | style='font-size:12.0pt;font-family:"Times New Roman","serif";mso-fareast-font-family:
| + | |
| - | "Times New Roman";mso-ansi-language:TR;mso-fareast-language:TR'>3.
| + | |
| - | Resuspend in 1/100 <span class=SpellE>volume</span> SGII+KNO3 <span
| + | |
| - | class=SpellE>and</span> <span class=SpellE>allow</span> <span
| + | |
| - | class=SpellE>to</span> <span class=SpellE>shake</span> at <span
| + | |
| - | class=SpellE>room</span> <span class=SpellE>temperature</span> <span
| + | |
| - | class=SpellE>for</span> 2-4 <span class=SpellE>hours</span>.<o:p></o:p></span></li>
| + | |
| - | <li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto; | + | |
| - | line-height:normal;mso-list:l1 level1 lfo1;tab-stops:list 36.0pt'><span
| + | |
| - | style='font-size:12.0pt;font-family:"Times New Roman","serif";mso-fareast-font-family:
| + | |
| - | "Times New Roman";mso-ansi-language:TR;mso-fareast-language:TR'>4. <span
| + | |
| - | class=SpellE>If</span> <span class=SpellE>desired</span>, <span
| + | |
| - | class=SpellE>treat</span> <span class=SpellE>cells</span> <span
| + | |
| - | class=SpellE>with</span> gamete <span class=SpellE>autolysin</span> (<span
| + | |
| - | class=SpellE>prepared</span> as <span class=SpellE>described</span> in <span
| + | |
| - | class=SpellE>The</span> Chlamydomonas <span class=SpellE>Sourcebook</span>*)
| + | |
| - | <span class=SpellE>for</span> <span class=SpellE>approximately</span> 45 <span
| + | |
| - | class=SpellE>minutes</span>. I <span class=SpellE>usually</span> resuspend
| + | |
| - | <span class=SpellE>the</span> <span class=SpellE>cells</span> in <span
| + | |
| - | class=SpellE>about</span> 1/25 <span class=SpellE>the</span> <span
| + | |
| - | class=SpellE>original</span> <span class=SpellE>volume</span> in <span
| + | |
| - | class=SpellE>autolysin</span>. <span class=SpellE>Spin</span> <span
| + | |
| - | class=SpellE>and</span> <span class=SpellE>wash</span> <span class=SpellE>once</span>
| + | |
| - | <span class=SpellE>with</span> SGII+KNO3 <span class=SpellE>before</span> <span
| + | |
| - | class=SpellE>transformation</span>. <span class=SpellE>Optional</span>:
| + | |
| - | Test <span class=SpellE>effectiveness</span> of lysis <span class=SpellE>by</span>
| + | |
| - | <span class=SpellE>sensitivity</span> <span class=SpellE>to</span> 0.05%
| + | |
| - | NP-40. (<span class=SpellE>Count</span> <span class=SpellE>duplicate</span>
| + | |
| - | <span class=SpellE>samples</span> +/- <span class=SpellE>detergent</span>
| + | |
| - | in <span class=SpellE>hemacytometer</span>.) <o:p></o:p></span></li>
| + | |
| - | </ul> | + | |
| | | | |
| - | </span> | + | <br>To make the final medium, mix the following: |
| | | | |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height: | + | <br> 2.42 g Tris |
| - | normal'><span class=SpellE><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
| + | <br> 25 ml solution #1 (salts) |
| - | mso-fareast-font-family:"Times New Roman";mso-ansi-language:TR;mso-fareast-language:
| + | <br> 0.375 ml solution #2 (phosphate) |
| - | TR'>Note</span></span><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
| + | <br> 1.0 ml solution #3 (trace elements) |
| - | mso-fareast-font-family:"Times New Roman";mso-ansi-language:TR;mso-fareast-language:
| + | <br> 1.0 ml glacial acetic acid |
| - | TR'>: <span class=SpellE>Several</span> <span class=SpellE>recent</span> <span
| + | <br> water to 1 liter |
| - | class=SpellE>experiments</span> <span class=SpellE>suggest</span> <span
| + | <br> |
| - | class=SpellE>that</span> <span class=SpellE>autolysin</span> <span
| + | <br> For solid medium, add 15 g agar per liter |
| - | class=SpellE>treatment</span> <span class=SpellE>increases</span> <span
| + | <br> |
| - | class=SpellE>the</span> <span class=SpellE>transformation</span> rate of <span
| + | <br>Autoclave. |
| - | class=SpellE>cw</span>-15 <span class=SpellE>mutants</span>.<o:p></o:p></span></p>
| + | |
| | | | |
| - | <ul type=disc> | + | <br>For Tris-minimal medium omit the acetic acid and titrate the final solution to pH 7.0 with HCl |
| - | <li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;
| + | <br> |
| - | line-height:normal;mso-list:l0 level1 lfo2;tab-stops:list 36.0pt'><span
| + | <br><b>Reference:</b><a href="http://www.chlamy.org/TAP.html">http://www.chlamy.org/TAP.html </a> |
| - | style='font-size:12.0pt;font-family:"Times New Roman","serif";mso-fareast-font-family:
| + | <br><br> |
| - | "Times New Roman";mso-ansi-language:TR;mso-fareast-language:TR'>5. <span
| + | |
| - | class=SpellE>Work</span> in <span class=SpellE>lots</span> of a <span
| + | |
| - | class=SpellE>few</span> <span class=SpellE>tubes</span> at a time. <span
| + | |
| - | class=SpellE>Add</span> 300 <span class=SpellE>microliters</span> <span
| + | |
| - | class=SpellE>cells</span>, 100 <span class=SpellE>microliters</span> 20% <span
| + | |
| - | class=SpellE>polyethylene</span> <span class=SpellE>glycol</span>, 1-2 <span
| + | |
| - | class=SpellE>micrograms</span> DNA (<span class=SpellE>linearized</span>
| + | |
| - | DNA <span class=SpellE>generally</span> <span class=SpellE>transforms</span>
| + | |
| - | <span class=SpellE>somewhat</span> <span class=SpellE>more</span> <span
| + | |
| - | class=SpellE>efficiently</span> <span class=SpellE>than</span> <span
| + | |
| - | class=SpellE>supercoiled</span>), <span class=SpellE>and</span> 300 mg <span
| + | |
| - | class=GramE>sterile</span> <span class=SpellE>glass</span>*** <span
| + | |
| - | class=SpellE>beads</span>. <span class=SpellE>Vortex</span> <span
| + | |
| - | class=SpellE>for</span> 15-30 <span class=SpellE>seconds</span> at top <span
| + | |
| - | class=SpellE>speed</span> on a <span class=SpellE>Fisher</span> <span
| + | |
| - | class=SpellE>Vortex</span> <span class=SpellE>Genie</span> 2 <span
| + | |
| - | class=SpellE>mixer</span>; <span class=SpellE>plate</span> <span
| + | |
| - | class=SpellE>cells</span> <span class=SpellE>immediately</span> on an
| + | |
| - | SGII+KNO3 <span class=SpellE>selective</span> <span class=SpellE>agar</span>**.
| + | |
| - | <span class=SpellE>Depending</span> on <span class=SpellE>age</span> <span
| + | |
| - | class=SpellE>and</span> <span class=SpellE>moisture</span> of <span
| + | |
| - | class=SpellE>plates</span>, <span class=SpellE>parafilm</span> <span
| + | |
| - | class=SpellE>immediately</span> <span class=SpellE>or</span> <span
| + | |
| - | class=SpellE>after</span> 24 <span class=SpellE>hours</span>; <span
| + | |
| - | class=SpellE>and</span> put in <span class=SpellE>the</span> <span
| + | |
| - | class=SpellE>light</span>.<o:p></o:p></span></li>
| + | |
| - | </ul>
| + | |
| | | | |
| - | <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height: | + | <br><b>Hutner's trace elements</b> |
| - | normal'><span class=SpellE><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
| + | <br> |
| - | mso-fareast-font-family:"Times New Roman";mso-ansi-language:TR;mso-fareast-language:
| + | <br>Hutner et al. (1950) Proc. Am. Philos. Soc. 94, 152-170 |
| - | TR'>Colonies</span></span><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
| + | <br> |
| - | mso-fareast-font-family:"Times New Roman";mso-ansi-language:TR;mso-fareast-language:
| + | <br>This mixture is used both in TAP and in the Sueoka high salt medium. |
| - | TR'> <span class=SpellE>should</span> be <span class=SpellE>visible</span> in <span
| + | <br> |
| - | class=SpellE>about</span> 6 <span class=SpellE>days</span>.<o:p></o:p></span></p>
| + | <br>For a detailed analysis of how well this trace elements solution meets the nutritional requirements of C. reinhardtii, <br>see Merchant et al. (2006) Biochim. Biophys. Acta 1763, 578-594. |
| | | | |
| - | <p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;
| + | |
| - | line-height:normal'><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
| + | |
| - | mso-fareast-font-family:"Times New Roman";mso-ansi-language:TR;mso-fareast-language:
| + | |
| - | TR'>* <span class=SpellE>The</span> <span class=SpellE>protocol</span> is <span
| + | |
| - | class=SpellE>followed</span> <span class=SpellE>except</span> <span
| + | |
| - | class=SpellE>that</span> <span class=SpellE>the</span> <span class=SpellE>culture</span>
| + | |
| - | <span class=SpellE>supernatant</span> of <span class=SpellE>the</span> <span
| + | |
| - | class=SpellE>mated</span> <span class=SpellE>gametes</span> is <span
| + | |
| - | class=SpellE>filtered</span> <span class=SpellE>through</span> a 0.22-0.45 <span
| + | |
| - | class=SpellE>micrometer</span> <span class=SpellE>nitrocellulose</span> <span
| + | |
| - | class=SpellE>filter</span>. I <span class=SpellE>don't</span> <span
| + | |
| - | class=SpellE>know</span> <span class=SpellE>how</span> <span class=SpellE>much</span>
| + | |
| - | of <span class=SpellE>the</span> <span class=SpellE>activity</span> (<span
| + | |
| - | class=SpellE>if</span> <span class=SpellE>any</span>) is <span class=SpellE>lost</span>
| + | |
| - | in <span class=SpellE>this</span> <span class=SpellE>protocol</span>, but <span
| + | |
| - | class=SpellE>the</span> <span class=SpellE>requirement</span> <span
| + | |
| - | class=SpellE>for</span> <span class=SpellE>sterility</span> <span class=SpellE>to</span>
| + | |
| - | <span class=SpellE>my</span> <span class=SpellE>mind</span> <span class=SpellE>outweighs</span>
| + | |
| - | <span class=SpellE>any</span> <span class=SpellE>loss</span> in <span
| + | |
| - | class=SpellE>this</span> step. <span class=SpellE>After</span> <span
| + | |
| - | class=SpellE>the</span> <span class=SpellE>filtration</span>, <span
| + | |
| - | class=SpellE>the</span> <span class=SpellE>supernatant</span> is <span
| + | |
| - | class=SpellE>frozen</span> in 50 ml <span class=SpellE>lots</span> at -20 <span
| + | |
| - | class=SpellE>deg</span> C.<o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto; | + | <br>For 1 liter final mix, dissolve each compound in the volume of water indicated. |
| - | line-height:normal'><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
| + | <br> |
| - | mso-fareast-font-family:"Times New Roman";mso-ansi-language:TR;mso-fareast-language:
| + | <br>The EDTA should be dissolved in boiling water, and the FeSO4 should be prepared last to avoid oxidation. |
| - | TR'>** <span class=SpellE>Use</span> <span class=SpellE>washed</span> <span
| + | <br>compound amount water |
| - | class=SpellE>agar</span> <span class=SpellE>to</span> <span class=SpellE>remove</span>
| + | <br>EDTA disodium salt 50 g 250 ml |
| - | <span class=SpellE>traces</span> of <span class=SpellE>ammonia</span> <span | + | <br>ZnSO4 . 7 H2O 22 g 100 ml |
| - | class=SpellE>and</span> <span class=SpellE>other</span> <span class=SpellE>impurities</span>.<o:p></o:p></span></p>
| + | <br>H3BO3 11.4 g 200 ml |
| | + | <br>MnCl2 . 4 H2O 5.06 g 50 ml |
| | + | <br>CoCl2. 6 H2O 1.61 g 50 ml |
| | + | <br>CuSO4 . 5 H2O 1.57 g 50 ml |
| | + | <br>(NH4)6Mo7O24. 4 H2O 1.10 g 50 ml |
| | + | <br>FeSO4. 7 H2O 4.99 g 50 ml |
| | + | <br> |
| | + | <br>Mix all solutions except EDTA. Bring to boil, then add EDTA solution. The mixture should turn green. When <br>everything is dissolved, cool to 70 degrees C. Keeping temperature at 70, add 85 ml hot 20% KOH solution (20 grams / 100 ml final volume).<br> Do NOT use NaOH to adjust the pH. |
| | + | <br> |
| | + | <br>Bring the final solution to 1 liter total volume. It should be clear green initially. Stopper the flask with a <br>cotton plug and let it stand for 1-2 weeks, shaking it once a day. The solution should eventually turn purple and <br>leave a rust-brown precipitate, which can be removed by filtering through two layers of Whatman#1 filter paper, <br>repeating the filtration if necessary until the solution is clear. Store refrigerated or frozen convenient <br>aliquots. Some people shorten the time for formation of the precipiate by bubbling the solution with filtered air. |
| | + | <br> |
| | + | <br>If no precipitate forms, the solution is still usable. However, you might want to check the pH in this case and <br>adjust it to around 7.0 using either KOH or HCl as needed. |
| | + | <br> |
| | + | <br>To prepare sulfur-free trace elements for hydrogen generation, the sulfate salts can be replaced with equimolar <br>chloride salts (ZnCl2 10.0 g; CuCl2 . 2 H2O 1.00 g; FeCl2 . 4 H2O, 3.60 g). . |
| | + | <br><b>Reference:</b><a href="http://www.chlamy.org/trace.html">http://www.chlamy.org/trace.html </a> |
| | + | <br> |
| | + | <br> |
| | + | <br><b>A. Glass bead method for nuclear transformation of <i>Chlamydomonas reinhardtii</i></b> |
| | + | <br><b>Materials.</b> |
| | + | <br>- Cell-wall deficient (e.g. cw15) host cell strain. (If you need to use a strain with a wild-type cell-wall, the <br>cells must be treated with autolysin prior to vortexing with glass beads (step 7).) |
| | + | <br>- Sterile liquid growth medium (permissive for the host cell line). (Approximately 35mL of culture / transformation <br>plate.) |
| | + | <br>- Sterile liquid growth medium (corresponding to selective conditions). (This will be used to wash the cells by <br>centrifugation before transformation. Use appropriate medium( minimal, arginine free, etc) depending on the <br>selection for transformants that will be applied.) |
| | + | <br>- Prepare glass tubes (3 mL) with 0.3g glass beads (Thomas Scientific), sterilize by baking in oven. (A convenient <br>scoop can be made from the bottom of an Eppendorf tube and a blue pipetman tip, glued by gently melting the tip). |
| | + | <br>- Sterile centrifugation bottles and tubes. |
| | + | <br>- Sterile, cotton-plugged 5 mL pipets. |
| | + | <br>- Plates with appropriate solid medium for selection of the transformants. |
| | + | <br>- DNA with selection marker. (Circular, supercoiled DNA can be used, but in cases where single insertions are <br>desirable (e.g. insertional mutagenesis), a linear DNA fragment is preferable. The amount of DNA used will also <br>influence the number of insertions (approx. range: 0.2 – 1.0 ug / transformation). |
| | + | <br> |
| | + | <br><b>Protocol.</b> |
| | + | <br>1. Grow cells in appropriate medium (permissive) to a density of ~2 x 106/ mL. |
| | + | <br>2. Collect cells by centrifugation in sterile centrifugation bottles at room temperature (3500 g x 10 min). Discard <br>supernatant. |
| | + | <br>3. Resuspend cells in 1/25 – 1/50 initial volume in selective medium with a cotton-plugged pipet. Transfer to a <br>sterile centrifugation tube. |
| | + | <br>4. Collect cells by centrifugation at room temperature (3500 g x 10 min). Discard supernatant |
| | + | <br>5. Resuspend cells at approximately in 1/70 initial volume in selective medium (approximately 3.0 x 108 cells / mL. |
| | + | <br>Count a 1/100 dilution with the hemacytometer under the microscope. Adjust the volume to obtain a concentration of <br>2. x 108 cells / mL. |
| | + | <br>6. To a tube containing 0.3g glass beads (sterilized by baking) add: |
| | + | <br>- 0.3 mL cell suspension. |
| | + | <br>- ~ 0.5 – 1.0 ug DNA. |
| | + | <br>7. Vortex at full speed for 15 seconds. |
| | + | <br> |
| | + | <br>8. Pour the contents of the tube on a selective plate, gently tilt and rotate the plate to spread the medium <br>evenly. |
| | + | <br>9. Allow the liquid to dry (protect from light to avoid phototactic movements of the cells). Seal the plates with <br>Parafilm (Micropore tape for minimal medium), and incubate under appropriate conditions for selection. (If cells <br>were grown under auxotrophic conditions (acetate, dark) and will be selected for photoautotrophic growth (minimal, <br>light), put the plates in dim light for 16 – 24 hours before transferring to light. Colonies will appear within 1-3 <br>weeks depending on the selection applied.) |
| | + | <br><b>References:</b> |
| | + | <br>Kindle, K. (1990) High-frequency nuclear transformation of <i>Chlamydomonas reinhardtii</i>. Proc. Natl. Acad. USA 87: <br>1228-1232. |
| | + | <br><br><br><b>B. Electroporation method for nuclear transformation of <i>Chlamydomonas reinhardtii</i></b> |
| | + | <br><b>Materials.</b> |
| | + | <br>- Cell-wall deficient host cell strain |
| | + | <br>- Sterile centrifugation bottles and tubes |
| | + | <br>- Electroporation cuvettes |
| | + | <br>- Plates with appropriate solid medium for selection of the transformants |
| | + | <br>- DNA with selection marker |
| | + | <br>- TAP, 40mM sucrose |
| | + | <br>- TAP, 40mM sucrose, 0.4% PEG 8 000 - Starch 20% |
| | + | <br>Starch 20% preparation |
| | + | <br>20 g starch in a centrifuge tube |
| | + | <br>Wash with ethanol 100% |
| | + | <br>Wash with water |
| | + | <br>Repeat 2 times |
| | + | <br>Resuspend in 100 ml Ethanol 70% |
| | + | <br>Aliquots of 20 ml and keep at room temperature |
| | + | <br>The day of transformation, centrifuge an aliquot 1 minute at 1 000 rpm |
| | + | <br>Wash 4 times with TAP + sucrose 40 mM |
| | + | <br>Resuspend in 20 ml of TAP + sucrose 40 mM + PEG 8 000 0.4% |
| | + | <br><b>Protocol.</b> |
| | + | <br>1. Grow 250 ml of cells to a density of 2 x 106 cells/ml |
| | + | <br>2. Collect cells by centrifugation at room temperature at 3 500 rpm for 5 minutes in sterile centrifugation <br>bottles. Discard supernatant |
| | + | <br> |
| | + | <br>3. Resuspend in 1.25 ml of TAP, 40mM sucrose |
| | + | <br>4. Incubate on ice 10 minutes |
| | + | <br>5. Transfer 250 μl of cells in a cuvette containing 1 μg of DNA |
| | + | <br>6. Incubate at room temperature 5 minutes |
| | + | <br>7. Electroporate 0.75 kV, 25 μF, no R, 6 msec |
| | + | <br>8. Incubate at room temperature 10 minutes |
| | + | <br>9. Add 1 ml of starch 20% and pour the contents of the cuvette on a selective plate, gently tilt and rotate the <br>plate to spread the medium |
| | + | <br>10. Allow the liquid to dry (protect from light), seal the plates with parafilm and incubate under appropriate <br>conditions for selection of transformants. |
| | + | <br> |
| | + | <br><b>References.</b> |
| | + | <br>Boynton et al (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240: <br>1534-1538. |
| | + | <br>Finer et al. (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Reports 11: <br>323-328. |
| | + | <br> |
| | | | |
| - | <p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;
| |
| - | line-height:normal'><span style='font-size:12.0pt;font-family:"Times New Roman","serif";
| |
| - | mso-fareast-font-family:"Times New Roman";mso-ansi-language:TR;mso-fareast-language:
| |
| - | TR'>***<span class=SpellE>Glass</span> <span class=SpellE>beads</span> <span
| |
| - | class=SpellE>are</span> <span class=SpellE>approximately</span> <span
| |
| - | class=GramE>0.5</span> mm in <span class=SpellE>diameter</span> <span
| |
| - | class=SpellE>and</span> <span class=SpellE>are</span> <span class=SpellE>available</span>
| |
| - | <span class=SpellE>from</span> Thomas. <span class=SpellE>We</span> <span
| |
| - | class=SpellE>wash</span> <span class=SpellE>them</span> <span class=SpellE>with</span>
| |
| - | <span class=SpellE>acid</span>, <span class=SpellE>rinse</span> <span
| |
| - | class=SpellE>them</span> <span class=SpellE>with</span> <span class=SpellE>water</span>
| |
| - | <span class=SpellE>until</span> <span class=SpellE>the</span> <span
| |
| - | class=SpellE>pH</span> is <span class=SpellE>neutral</span>, <span
| |
| - | class=SpellE>dry</span> <span class=SpellE>them</span>, <span class=SpellE>and</span>
| |
| - | <span class=SpellE>then</span> <span class=SpellE>weigh</span> 300 mg <span
| |
| - | class=SpellE>into</span> <span class=SpellE>glass</span> <span class=SpellE>culture</span>
| |
| - | <span class=SpellE>tubes</span> <span class=SpellE>and</span> <span
| |
| - | class=SpellE>bake</span> <span class=SpellE>for</span> <span class=SpellE>approximately</span>
| |
| - | 2 <span class=SpellE>hours</span> at 400 <span class=SpellE>deg</span> F.<o:p></o:p></span></p>
| |
| | | | |
| - | <p class=MsoNormal><span lang=EN-US><o:p> </o:p></span></p>
| |
| - | <div class=WordSection1>
| |
| | | | |
| - | <p class=Default><span class=SpellE><b><span style='font-size:11.5pt;
| |
| - | color:windowtext'>Different Protocol to Transform Chlamy with Glass Beads</span></b></span><b><span style='font-size:11.5pt;
| |
| - | color:windowtext'>. </span></b><span style='font-size:11.5pt;color:windowtext'><o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>1. <span
| |
| - | class=SpellE>Grow</span> <span class=SpellE>cells</span> in <span class=SpellE>appropriate</span>
| |
| - | <span class=SpellE>medium</span> (<span class=SpellE>permissive</span>) <span
| |
| - | class=SpellE>to</span> a <span class=SpellE>density</span> of ~2 x 10<sup><span
| |
| - | style='position:relative;top:-5.0pt;mso-text-raise:5.0pt'>6</span></sup>/ mL. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>2. <span
| |
| - | class=SpellE>Collect</span> <span class=SpellE>cells</span> <span class=SpellE>by</span>
| |
| - | <span class=SpellE>centrifugation</span> in <span class=GramE>sterile</span> <span
| |
| - | class=SpellE>centrifugation</span> <span class=SpellE>bottles</span> at <span
| |
| - | class=SpellE>room</span> <span class=SpellE>temperature</span> (3500 g x 10 <span
| |
| - | class=SpellE>min</span>). <span class=SpellE>Discard</span> <span class=SpellE>supernatant</span>.
| |
| - | <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>3. Resuspend <span
| |
| - | class=SpellE>cells</span> in 1/25 – 1/50 <span class=SpellE>initial</span> <span
| |
| - | class=SpellE>volume</span> in <span class=SpellE>selective</span> <span
| |
| - | class=SpellE>medium</span> <span class=SpellE>with</span> a <span class=SpellE>cotton</span>-<span
| |
| - | class=SpellE>plugged</span> pipet. Transfer <span class=SpellE>to</span> a <span
| |
| - | class=GramE>sterile</span> <span class=SpellE>centrifugation</span> <span
| |
| - | class=SpellE>tube</span>. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>4. <span
| |
| - | class=SpellE>Collect</span> <span class=SpellE>cells</span> <span class=SpellE>by</span>
| |
| - | <span class=SpellE>centrifugation</span> at <span class=SpellE>room</span> <span
| |
| - | class=SpellE>temperature</span> (3500 g x 10 <span class=SpellE>min</span>). <span
| |
| - | class=SpellE>Discard</span> <span class=SpellE>supernatant</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>5. Resuspend <span
| |
| - | class=SpellE>cells</span> at <span class=SpellE>approximately</span> in 1/70 <span
| |
| - | class=SpellE>initial</span> <span class=SpellE>volume</span> in <span
| |
| - | class=SpellE>selective</span> <span class=SpellE>medium</span> (<span
| |
| - | class=SpellE>approximately</span> <span class=GramE>3.0</span> x 10<sup><span
| |
| - | style='position:relative;top:-5.0pt;mso-text-raise:5.0pt'>8 </span></sup><span
| |
| - | class=SpellE>cells</span> / mL. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><span style='font-size:11.5pt;color:windowtext'>Count</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'> a 1/100 <span class=SpellE>dilution</span>
| |
| - | <span class=SpellE>with</span> <span class=SpellE>the</span> <span
| |
| - | class=SpellE>hemacytometer</span> <span class=SpellE>under</span> <span
| |
| - | class=SpellE>the</span> <span class=SpellE>microscope</span>. <span
| |
| - | class=SpellE>Adjust</span> <span class=SpellE>the</span> <span class=SpellE>volume</span>
| |
| - | <span class=SpellE>to</span> <span class=SpellE>obtain</span> a <span
| |
| - | class=SpellE>concentration</span> of 2. x 10<sup><span style='position:relative;
| |
| - | top:-5.0pt;mso-text-raise:5.0pt'>8 </span></sup><span class=SpellE>cells</span>
| |
| - | / mL. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>6. <span
| |
| - | class=SpellE>To</span> a <span class=SpellE>tube</span> <span class=SpellE>containing</span>
| |
| - | 0.3g <span class=SpellE>glass</span> <span class=SpellE>beads</span> (<span
| |
| - | class=SpellE>sterilized</span> <span class=SpellE>by</span> <span class=SpellE>baking</span>)
| |
| - | <span class=SpellE>add</span>: <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default style='text-indent:36.0pt'><span style='font-size:11.5pt;
| |
| - | color:windowtext'>- 0.3 mL <span class=SpellE>cell</span> <span class=SpellE>suspension</span>.
| |
| - | <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- ~ 0.5 – <span
| |
| - | class=GramE>1.0</span> <span class=SpellE>ug</span> DNA. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>7. <span
| |
| - | class=SpellE>Vortex</span> at <span class=SpellE>full</span> <span
| |
| - | class=SpellE>speed</span> <span class=SpellE>for</span> 15 <span class=SpellE>seconds</span>.
| |
| - | <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='color:windowtext'><o:p> </o:p></span></p>
| |
| | | | |
| - | <p class=Default style='page-break-before:always'><span style='font-size:11.5pt;
| |
| - | color:windowtext'>8. <span class=SpellE>Pour</span> <span class=SpellE>the</span>
| |
| - | <span class=SpellE>contents</span> of <span class=SpellE>the</span> <span
| |
| - | class=SpellE>tube</span> on a <span class=SpellE>selective</span> <span
| |
| - | class=SpellE>plate</span>, <span class=SpellE>gently</span> <span class=SpellE>tilt</span>
| |
| - | <span class=SpellE>and</span> <span class=SpellE>rotate</span> <span
| |
| - | class=SpellE>the</span> <span class=SpellE>plate</span> <span class=SpellE>to</span>
| |
| - | <span class=GramE>spread</span> <span class=SpellE>the</span> <span
| |
| - | class=SpellE>medium</span> <span class=SpellE>evenly</span>. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>9. <span
| |
| - | class=SpellE>Allow</span> <span class=SpellE>the</span> <span class=SpellE>liquid</span>
| |
| - | <span class=SpellE>to</span> <span class=SpellE>dry</span> (<span class=SpellE>protect</span>
| |
| - | <span class=SpellE>from</span> <span class=SpellE>light</span> <span
| |
| - | class=SpellE>to</span> <span class=SpellE>avoid</span> <span class=SpellE>phototactic</span>
| |
| - | <span class=SpellE>movements</span> of <span class=SpellE>the</span> <span
| |
| - | class=SpellE>cells</span>). <span class=SpellE>Seal</span> <span class=SpellE>the</span>
| |
| - | <span class=SpellE>plates</span> <span class=SpellE>with</span> <span
| |
| - | class=SpellE>Parafilm</span> (<span class=SpellE>Micropore</span> <span
| |
| - | class=SpellE>tape</span> <span class=SpellE>for</span> minimal <span
| |
| - | class=SpellE>medium</span>), <span class=SpellE>and</span> <span class=SpellE>incubate</span>
| |
| - | <span class=SpellE>under</span> <span class=SpellE>appropriate</span> <span
| |
| - | class=SpellE>conditions</span> <span class=SpellE>for</span> <span
| |
| - | class=SpellE>selection</span>. <span class=GramE>(</span><span class=SpellE><i>If</i></span><i>
| |
| - | <span class=SpellE>cells</span> <span class=SpellE>were</span> <span
| |
| - | class=SpellE>grown</span> <span class=SpellE>under</span> <span class=SpellE>auxotrophic</span>
| |
| - | <span class=SpellE>conditions</span> (<span class=SpellE>acetate</span>, <span
| |
| - | class=SpellE>dark</span>) <span class=SpellE>and</span> <span class=SpellE>will</span>
| |
| - | be <span class=SpellE>selected</span> <span class=SpellE>for</span> <span
| |
| - | class=SpellE>photoautotrophic</span> <span class=SpellE>growth</span> (minimal,
| |
| - | <span class=SpellE>light</span></i>)<i>, put <span class=SpellE>the</span> <span
| |
| - | class=SpellE>plates</span> in <span class=SpellE>dim</span> <span class=SpellE>light</span>
| |
| - | <span class=SpellE>for</span> 16 – 24 <span class=SpellE>hours</span> <span
| |
| - | class=SpellE>before</span> <span class=SpellE>transferring</span> <span
| |
| - | class=SpellE>to</span> <span class=SpellE>light</span>. <span class=SpellE>Colonies</span>
| |
| - | <span class=SpellE>will</span> <span class=SpellE>appear</span> <span
| |
| - | class=SpellE>within</span> 1-3 <span class=SpellE>weeks</span> <span
| |
| - | class=SpellE>depending</span> on <span class=SpellE>the</span> <span
| |
| - | class=SpellE>selection</span> <span class=SpellE>applied</span>.<span
| |
| - | class=GramE>)</span> </i><o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><b><span style='font-size:11.5pt;
| |
| - | color:windowtext'>Reference</span></b><span style='font-size:11.5pt;color:windowtext'>s</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'>: <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default style='text-indent:36.0pt'><span class=SpellE><span
| |
| - | style='font-size:11.5pt;color:windowtext'>Kindle</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'>, K. (1990) <span class=SpellE>High</span>-<span
| |
| - | class=SpellE>frequency</span> <span class=SpellE>nuclear</span> <span
| |
| - | class=SpellE>transformation</span> of <i>Chlamydomonas reinhardtii</i>. <span
| |
| - | class=SpellE>Proc</span>. <span class=SpellE>Natl</span>. <span class=SpellE>Acad</span>.
| |
| - | USA <b>87</b>: 1228-1232. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><b><span style='font-size:14.0pt;color:windowtext'>B. <span
| |
| - | class=SpellE>Electroporation</span> <span class=SpellE>method</span> <span
| |
| - | class=SpellE>for</span> <span class=SpellE>nuclear</span> <span class=SpellE>transformation</span>
| |
| - | of <i>Chlamydomonas reinhard</i></span></b><b><i><span style='font-size:11.5pt;
| |
| - | color:windowtext'>tii </span></i></b><span style='font-size:11.5pt;color:windowtext'><o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><b><span style='font-size:11.5pt;
| |
| - | color:windowtext'>Materials</span></b></span><b><span style='font-size:11.5pt;
| |
| - | color:windowtext'> </span></b><span style='font-size:11.5pt;color:windowtext'><o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- <span
| |
| - | class=SpellE>Cell</span>-<span class=SpellE>wall</span> <span class=SpellE>deficient</span>
| |
| - | <span class=SpellE>host</span> <span class=SpellE>cell</span> <span
| |
| - | class=SpellE>strain</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- Sterile <span
| |
| - | class=SpellE>centrifugation</span> <span class=SpellE>bottles</span> <span
| |
| - | class=SpellE>and</span> <span class=SpellE>tubes</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- <span | + | <!-- up to here --> |
| - | class=SpellE>Electroporation</span> <span class=SpellE>cuvettes</span> <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- <span
| |
| - | class=SpellE>Plates</span> <span class=SpellE>with</span> <span class=SpellE>appropriate</span>
| |
| - | <span class=SpellE>solid</span> <span class=SpellE>medium</span> <span
| |
| - | class=SpellE>for</span> <span class=SpellE>selection</span> of <span
| |
| - | class=SpellE>the</span> <span class=SpellE>transformants</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- DNA <span
| |
| - | class=SpellE>with</span> <span class=SpellE>selection</span> marker <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- TAP, 40mM <span | + | </div> |
| - | class=SpellE>sucrose</span> <o:p></o:p></span></p>
| + | <div class='popingProblem' id='popingProblem' >There is a problem with poping up if you see this note.</div> <!-- dont delete this line, it is mandatory for poping up --> |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- TAP, 40mM <span
| |
| - | class=SpellE>sucrose</span>, <span class=GramE>0.4</span>% PEG 8 000 - <span
| |
| - | class=SpellE>Starch</span> 20% <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><b><span style='font-size:11.5pt;
| |
| - | color:windowtext'>Starch</span></b></span><b><span style='font-size:11.5pt;
| |
| - | color:windowtext'> 20% <span class=SpellE>preparation</span> </span></b><span
| |
| - | style='font-size:11.5pt;color:windowtext'><o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>20 g <span
| |
| - | class=SpellE>starch</span> in a <span class=SpellE>centrifuge</span> <span
| |
| - | class=SpellE>tube</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><span style='font-size:11.5pt;color:windowtext'>Wash</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'> <span class=SpellE>with</span> <span
| |
| - | class=SpellE>ethanol</span> 100% <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><span style='font-size:11.5pt;color:windowtext'>Wash</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'> <span class=SpellE>with</span> <span
| |
| - | class=SpellE>water</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><span style='font-size:11.5pt;color:windowtext'>Repeat</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'> 2 <span class=SpellE>times</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>Resuspend in
| |
| - | 100 ml <span class=SpellE>Ethanol</span> 70% <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><span style='font-size:11.5pt;color:windowtext'>Aliquots</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'> of 20 ml <span class=SpellE>and</span>
| |
| - | <span class=SpellE>keep</span> at <span class=SpellE>room</span> <span
| |
| - | class=SpellE>temperature</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><span style='font-size:11.5pt;color:windowtext'>The</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'> <span class=SpellE>day</span> of <span
| |
| - | class=SpellE>transformation</span>, <span class=SpellE>centrifuge</span> an <span
| |
| - | class=SpellE>aliquot</span> 1 <span class=SpellE>minute</span> at 1 000 <span
| |
| - | class=SpellE>rpm</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span class=SpellE><span style='font-size:11.5pt;color:windowtext'>Wash</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'> 4 <span class=SpellE>times</span> <span
| |
| - | class=SpellE>with</span> TAP + <span class=SpellE>sucrose</span> 40 mM <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>Resuspend in
| |
| - | 20 ml of TAP + <span class=SpellE>sucrose</span> 40 mM + PEG 8 000 <span
| |
| - | class=GramE>0.4</span>% <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><b><span style='font-size:11.5pt;color:windowtext'>Protocol </span></b><span | + | <script type="text/javascript"> |
| - | style='font-size:11.5pt;color:windowtext'><o:p></o:p></span></p>
| + | for(var i =0;i<list.length;i++){ |
| | + | var temp=list[i]; |
| | + | $("div.slide_content").html($("div.slide_content").html().replace( temp, "<a class=\"anchor\" href=\"https://2011.igem.org/Team:Bilkent_UNAM_Turkey/"+temp+"\" id=\"searchlink"+i+"\" rel=\"subcontent"+i+"\" rev=\"https://2011.igem.org/Team:Bilkent_UNAM_Turkey/pops/"+temp+"?action=raw&ctype=text/css\">"+temp+"</a>" )); |
| | + | |
| | + | } |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>1. <span
| |
| - | class=SpellE>Grow</span> 250 ml of <span class=SpellE>cells</span> <span
| |
| - | class=SpellE>to</span> a <span class=SpellE>density</span> of 2 x 10<sup><span
| |
| - | style='position:relative;top:-5.0pt;mso-text-raise:5.0pt'>6 </span></sup><span
| |
| - | class=SpellE>cells</span>/ml <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>2. <span
| + | //this is for banner |
| - | class=SpellE>Collect</span> <span class=SpellE>cells</span> <span class=SpellE>by</span>
| + | |
| - | <span class=SpellE>centrifugation</span> at <span class=SpellE>room</span> <span
| + | |
| - | class=SpellE>temperature</span> at 3 500 <span class=SpellE>rpm</span> <span
| + | |
| - | class=SpellE>for</span> 5 <span class=SpellE>minutes</span> in <span
| + | |
| - | class=GramE>sterile</span> <span class=SpellE>centrifugation</span> <span
| + | |
| - | class=SpellE>bottles</span>. <span class=SpellE>Discard</span> <span
| + | |
| - | class=SpellE>supernatant</span> <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default><a name="_GoBack"></a><span style='color:windowtext'><o:p> </o:p></span></p>
| |
| | | | |
| - | <p class=Default style='page-break-before:always'><span style='font-size:11.5pt;
| + | var tr1 = document.createElement('tr'); |
| - | color:windowtext'>3. Resuspend in 1.25 ml of TAP, 40mM <span class=SpellE>sucrose</span>
| + | var table1 = document.createElement('table'); |
| - | <o:p></o:p></span></p>
| + | table1.setAttribute('id','banner'); |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>4. <span
| |
| - | class=SpellE>Incubate</span> on <span class=SpellE>ice</span> 10 <span
| |
| - | class=SpellE>minutes</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>5. Transfer
| + | var image1 = document.createElement('img'); |
| - | 250 <span class=SpellE>μl</span> of <span class=SpellE>cells</span> in a <span
| + | image1.setAttribute('src', 'https://static.igem.org/mediawiki/2011/a/a1/Unam.gif'); |
| - | class=SpellE>cuvette</span> <span class=SpellE>containing</span> 1 <span
| + | image1.setAttribute('style','border:0;'); |
| - | class=SpellE>μg</span> of DNA <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>6. <span
| + | var image2 = document.createElement('img'); |
| - | class=SpellE>Incubate</span> at <span class=SpellE>room</span> <span
| + | image2.setAttribute('src', 'https://static.igem.org/mediawiki/2011/0/05/Igem.gif'); |
| - | class=SpellE>temperature</span> 5 <span class=SpellE>minutes</span> <o:p></o:p></span></p>
| + | image2.setAttribute('style','border:0;'); |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>7. <span
| + | var image3 = document.createElement('img'); |
| - | class=SpellE>Electroporate</span> 0.75 <span class=SpellE>kV</span>, 25
| + | image3.setAttribute('src', 'https://static.igem.org/mediawiki/2011/0/06/But.gif'); |
| - | μF, no R, 6 <span class=SpellE>msec</span> <o:p></o:p></span></p>
| + | image3.setAttribute('style','border:0;'); |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>8. <span
| + | var image4 = document.createElement('img'); |
| - | class=SpellE>Incubate</span> at <span class=SpellE>room</span> <span
| + | image4.setAttribute('src', 'https://static.igem.org/mediawiki/2011/3/32/Bilkent.gif'); |
| - | class=SpellE>temperature</span> 10 <span class=SpellE>minutes</span> <o:p></o:p></span></p>
| + | image4.setAttribute('style','border:0;'); |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>9. <span
| + | var href1 = document.createElement('a'); |
| - | class=SpellE>Add</span> 1 ml of <span class=SpellE>starch</span> 20% <span
| + | href1.setAttribute('href','http://www.nano.org.tr/index1.html'); |
| - | class=SpellE>and</span> <span class=SpellE>pour</span> <span class=SpellE>the</span>
| + | |
| - | <span class=SpellE>contents</span> of <span class=SpellE>the</span> <span
| + | |
| - | class=SpellE>cuvette</span> on a <span class=SpellE>selective</span> <span
| + | |
| - | class=SpellE>plate</span>, <span class=SpellE>gently</span> <span class=SpellE>tilt</span>
| + | |
| - | <span class=SpellE>and</span> <span class=SpellE>rotate</span> <span
| + | |
| - | class=SpellE>the</span> <span class=SpellE>plate</span> <span class=SpellE>to</span>
| + | |
| - | <span class=GramE>spread</span> <span class=SpellE>the</span> <span
| + | |
| - | class=SpellE>medium</span> <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>10. <span
| + | var href2 = document.createElement('a'); |
| - | class=SpellE>Allow</span> <span class=SpellE>the</span> <span class=SpellE>liquid</span>
| + | href2.setAttribute('href','https://2011.igem.org/Main_Page'); |
| - | <span class=SpellE>to</span> <span class=SpellE>dry</span> (<span class=SpellE>protect</span>
| + | |
| - | <span class=SpellE>from</span> <span class=SpellE>light</span>), <span
| + | |
| - | class=SpellE>seal</span> <span class=SpellE>the</span> <span class=SpellE>plates</span>
| + | |
| - | <span class=SpellE>with</span> <span class=SpellE>parafilm</span> <span
| + | |
| - | class=SpellE>and</span> <span class=SpellE>incubate</span> <span class=SpellE>under</span>
| + | |
| - | <span class=SpellE>appropriate</span> <span class=SpellE>conditions</span> <span
| + | |
| - | class=SpellE>for</span> <span class=SpellE>selection</span> of <span
| + | |
| - | class=SpellE>transformants</span>. <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default><b><span style='font-size:14.0pt;color:windowtext'>C. <span
| + | var href3 = document.createElement('a'); |
| - | class=SpellE>Chloroplast</span> <span class=SpellE>transformation</span> of <i>Chlamydomonas
| + | href3.setAttribute('href','https://2011.igem.org/Team:Bilkent_UNAM_Turkey'); |
| - | reinhardtii </i></span></b><span style='font-size:14.0pt;color:windowtext'><o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default><span class=SpellE><b><span style='font-size:11.5pt;
| + | var href4 = document.createElement('a'); |
| - | color:windowtext'>Materials</span></b></span><b><span style='font-size:11.5pt;
| + | href4.setAttribute('href','http://www.bilkent.edu.tr'); |
| - | color:windowtext'>. </span></b><span style='font-size:11.5pt;color:windowtext'><o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| + | var td1 = document.createElement('td'); |
| - | style='font-size:11.5pt;color:windowtext'>- <span class=SpellE>Host</span> <span
| + | var td2 = document.createElement('td'); |
| - | class=SpellE>cell</span> <span class=SpellE>strain</span>. <o:p></o:p></span></p>
| + | var td3 = document.createElement('td'); |
| | + | var td4 = document.createElement('td'); |
| | + | var td5 = document.createElement('td'); |
| | + | td5.setAttribute('width','110'); |
| | + | td5.appendChild(document.createElement('g:plusone')); |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| |
| - | style='font-size:11.5pt;color:windowtext'>- Sterile <span class=SpellE>liquid</span>
| |
| - | <span class=SpellE>growth</span> <span class=SpellE>medium</span> (<span
| |
| - | class=SpellE>permissive</span> <span class=SpellE>for</span> <span
| |
| - | class=SpellE>the</span> <span class=SpellE>host</span> <span class=SpellE>cell</span>
| |
| - | <span class=SpellE>line</span>). (<span class=SpellE><i>Approximately</i></span><i>
| |
| - | 10 mL of <span class=SpellE>culture</span> / <span class=SpellE>transformation</span>
| |
| - | <span class=SpellE>plate</span>.</i>) <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| + | href1.appendChild(image1); |
| - | style='font-size:11.5pt;color:windowtext'>- Sterile <span class=SpellE>liquid</span>
| + | href2.appendChild(image2); |
| - | <span class=SpellE>growth</span> <span class=SpellE>medium</span> (<span
| + | href3.appendChild(image3); |
| - | class=SpellE>corresponding</span> <span class=SpellE>to</span> <span
| + | href4.appendChild(image4); |
| - | class=SpellE>selective</span> <span class=SpellE>conditions</span>). <span
| + | |
| - | class=GramE>(</span><span class=SpellE><i>This</i></span><i> <span
| + | |
| - | class=SpellE>will</span> be <span class=SpellE>used</span> <span class=SpellE>to</span>
| + | |
| - | <span class=SpellE>wash</span> <span class=SpellE>the</span> <span
| + | |
| - | class=SpellE>cells</span> <span class=SpellE>by</span> <span class=SpellE>centrifugation</span>
| + | |
| - | <span class=SpellE>before</span> <span class=SpellE>transformation</span>. <span
| + | |
| - | class=SpellE>Use</span> <span class=SpellE>appropriate</span> <span
| + | |
| - | class=SpellE>medium</span>(<span class=SpellE>eg</span> minimal) <span
| + | |
| - | class=SpellE>depending</span> on <span class=SpellE>the</span> <span
| + | |
| - | class=SpellE>selection</span> <span class=SpellE>for</span> <span class=SpellE>transformants</span>
| + | |
| - | <span class=SpellE>that</span> <span class=SpellE>will</span> be <span
| + | |
| - | class=SpellE>applied</span>.</i><span class=GramE>)</span> <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| + | td1.appendChild(href1); |
| - | style='font-size:11.5pt;color:windowtext'>- Sterile <span class=SpellE>centrifugation</span>
| + | td2.appendChild(href2); |
| - | <span class=SpellE>bottles</span> <span class=SpellE>and</span> <span
| + | td3.appendChild(href3); |
| - | class=SpellE>tubes</span>. <o:p></o:p></span></p>
| + | td4.appendChild(href4); |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| |
| - | style='font-size:11.5pt;color:windowtext'>- Sterile, <span class=SpellE>cotton</span>-<span
| |
| - | class=SpellE>plugged</span> 5 mL <span class=SpellE>pipets</span>. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| + | tr1.appendChild(td1); |
| - | style='font-size:11.5pt;color:windowtext'>- <span class=SpellE>Plates</span> <span
| + | tr1.appendChild(td5); |
| - | class=SpellE>with</span> <span class=SpellE>appropriate</span> <span
| + | tr1.appendChild(td2); |
| - | class=SpellE>solid</span> <span class=SpellE>medium</span> <span class=SpellE>for</span>
| + | tr1.appendChild(td3); |
| - | <span class=SpellE>selection</span> of <span class=SpellE>the</span> <span
| + | tr1.appendChild(td4); |
| - | class=SpellE>transformants</span>. <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| + | table1.appendChild(tr1); |
| - | style='font-size:11.5pt;color:windowtext'>- DNA <span class=SpellE>with</span> <span
| + | |
| - | class=SpellE>selection</span> marker. (<i>1ug / uL, <span class=GramE>1.0</span>
| + | |
| - | <span class=SpellE>ug</span> <span class=SpellE>per</span> <span class=SpellE>sample</span>,
| + | |
| - | <span class=SpellE>sufficient</span> <span class=SpellE>for</span> <span
| + | |
| - | class=SpellE>up</span> <span class=SpellE>to</span> 7 <span class=SpellE>plates</span></i>).
| + | |
| - | <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| |
| - | style='font-size:11.5pt;color:windowtext'>- 100 mg/mL tungsten <span
| |
| - | class=SpellE>powder</span> in <span class=GramE>sterile</span> 50 % <span
| |
| - | class=SpellE>glycerol</span>. (<i>25 uL <span class=SpellE>per</span> <span
| |
| - | class=SpellE>sample</span></i>) <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| + | document.getElementById('top-section').appendChild(table1); |
| - | style='font-size:11.5pt;color:windowtext'>- 2 M <span class=SpellE>CaCl</span><sub><span
| + | |
| - | style='position:relative;top:5.0pt;mso-text-raise:-5.0pt'>2</span></sub>, <span
| + | |
| - | class=GramE>sterile</span> (<i>25 uL <span class=SpellE>per</span> <span
| + | |
| - | class=SpellE>sample</span></i>) <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| |
| - | style='font-size:11.5pt;color:windowtext'>- 100mM <span class=SpellE>spermidine</span>
| |
| - | (<span class=SpellE>base</span>), <span class=SpellE>filter</span> <span
| |
| - | class=SpellE>sterilized</span>. (<i>10 uL <span class=SpellE>per</span> <span
| |
| - | class=SpellE>sample</span></i>) <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| + | //banner up to here |
| - | style='font-size:11.5pt;color:windowtext'>- <span class=SpellE>Filter</span> <span
| + | |
| - | class=SpellE>holders</span> <span class=SpellE>for</span> <span class=SpellE>Helium</span>
| + | |
| - | <span class=SpellE>gun</span>.(<i>Sterilize <span class=SpellE>by</span> <span
| + | |
| - | class=SpellE>washing</span> <span class=SpellE>with</span> <span class=SpellE>Ethanol</span>,
| + | |
| - | <span class=SpellE>air</span> <span class=SpellE>dry</span> in <span
| + | |
| - | class=GramE>sterile</span> <span class=SpellE>hood</span></i>) <o:p></o:p></span></p>
| + | |
| | | | |
| - | <p class=Default style='margin-left:18.0pt;text-indent:-18.0pt'><span
| |
| - | style='font-size:11.5pt;color:windowtext'>- Sterile <span class=SpellE>microfuge</span>
| |
| - | <span class=SpellE>tubes</span> <span class=SpellE>and</span> <span
| |
| - | class=SpellE>tips</span>. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'><o:p> </o:p></span></p>
| |
| | | | |
| - | <p class=Default><b><span style='font-size:11.5pt;color:windowtext'>Protocol. </span></b><span
| |
| - | style='font-size:11.5pt;color:windowtext'><o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>1. <span
| |
| - | class=SpellE>Grow</span> <span class=SpellE>cells</span> in <span class=SpellE>appropriate</span>
| |
| - | <span class=SpellE>medium</span> (<span class=SpellE>permissive</span>) <span
| |
| - | class=SpellE>to</span> a <span class=SpellE>density</span> of ~2 x 10<sup><span
| |
| - | style='position:relative;top:-5.0pt;mso-text-raise:5.0pt'>6</span></sup>/ mL. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>2. <span
| |
| - | class=SpellE>Collect</span> <span class=SpellE>cells</span> <span class=SpellE>by</span>
| |
| - | <span class=SpellE>centrifugation</span> in <span class=GramE>sterile</span> <span
| |
| - | class=SpellE>centrifugation</span> <span class=SpellE>bottles</span> at <span
| |
| - | class=SpellE>room</span> <span class=SpellE>temperature</span> (3500 g x 10 <span
| |
| - | class=SpellE>min</span>). <span class=SpellE>Discard</span> <span class=SpellE>supernatant</span>.
| |
| - | <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='color:windowtext'>12 <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default style='page-break-before:always'><span style='font-size:11.5pt;
| |
| - | color:windowtext'>3. Resuspend <span class=SpellE>cells</span> in 1/30 <span
| |
| - | class=SpellE>initial</span> <span class=SpellE>volume</span> in <span
| |
| - | class=SpellE>selective</span> <span class=SpellE>medium</span> <span
| |
| - | class=SpellE>with</span> a <span class=SpellE>cotton</span>-<span class=SpellE>plugged</span>
| |
| - | pipet. Transfer <span class=SpellE>to</span> a <span class=GramE>sterile</span>
| |
| - | <span class=SpellE>centrifugation</span> <span class=SpellE>tube</span>. (<span
| |
| - | class=SpellE><i>Steps</i></span><i> 3 <span class=SpellE>and</span> 4 can be <span
| |
| - | class=SpellE>omitted</span> <span class=SpellE>if</span> <span class=SpellE>the</span>
| |
| - | <span class=SpellE>media</span> <span class=SpellE>for</span> <span
| |
| - | class=SpellE>the</span> <span class=SpellE>culture</span> <span class=SpellE>and</span>
| |
| - | <span class=SpellE>for</span> <span class=SpellE>selection</span> on <span
| |
| - | class=SpellE>the</span> <span class=SpellE>plates</span> <span class=SpellE>are</span>
| |
| - | <span class=SpellE>compatible</span></i>). <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>4. <span
| |
| - | class=SpellE>Collect</span> <span class=SpellE>cells</span> <span class=SpellE>by</span>
| |
| - | <span class=SpellE>centrifugation</span> at <span class=SpellE>room</span> <span
| |
| - | class=SpellE>temperature</span> (3500 g x 10 <span class=SpellE>min</span>). <span
| |
| - | class=SpellE>Discard</span> <span class=SpellE>supernatant</span> <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>5. Resuspend <span
| |
| - | class=SpellE>cells</span> in 1/30 <span class=SpellE>initial</span> <span
| |
| - | class=SpellE>volume</span> in <span class=SpellE>selective</span> <span
| |
| - | class=SpellE>medium</span> (<span class=SpellE>approximately</span> 6 x 10<sup><span
| |
| - | style='position:relative;top:-5.0pt;mso-text-raise:5.0pt'>7 </span></sup><span
| |
| - | class=SpellE>cells</span> / mL). <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>6. <span
| |
| - | class=SpellE>Plate</span> <span class=GramE>0.3</span> mL of <span
| |
| - | class=SpellE>cell</span> <span class=SpellE>suspension</span> <span
| |
| - | class=SpellE>evenly</span> on <span class=SpellE>selective</span> <span
| |
| - | class=SpellE>plate</span>. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>7. <span
| |
| - | class=SpellE>Allow</span> <span class=SpellE>the</span> <span class=SpellE>liquid</span>
| |
| - | <span class=SpellE>to</span> <span class=SpellE>dry</span> (<span class=SpellE>protect</span>
| |
| - | <span class=SpellE>from</span> <span class=SpellE>light</span> <span
| |
| - | class=SpellE>to</span> <span class=SpellE>avoid</span> <span class=SpellE>phototactic</span>
| |
| - | <span class=SpellE>movements</span> of <span class=SpellE>the</span> <span
| |
| - | class=SpellE>cells</span>). <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>8. <span
| |
| - | class=SpellE>Sonicate</span> <span class=SpellE>the</span> tungsten <span
| |
| - | class=SpellE>suspension</span> <span class=SpellE>briefly</span> (<span
| |
| - | class=SpellE>the</span> <span class=SpellE>tube</span> is <span class=SpellE>attached</span>
| |
| - | <span class=SpellE>with</span> a <span class=SpellE>stand</span> <span
| |
| - | class=SpellE>and</span> <span class=SpellE>clamp</span> <span class=SpellE>so</span>
| |
| - | as <span class=SpellE>to</span> <span class=SpellE>touch</span> <span
| |
| - | class=SpellE>the</span> tip of <span class=SpellE>the</span> <span
| |
| - | class=SpellE>sonication</span> <span class=SpellE>probe</span> <span
| |
| - | class=SpellE>immersed</span> in a <span class=SpellE>beaker</span> of <span
| |
| - | class=SpellE>water</span>). <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>9) <span
| |
| - | class=SpellE>In</span> a <span class=GramE>sterile</span> <span class=SpellE>microfuge</span>
| |
| - | <span class=SpellE>tube</span> <span class=SpellE>placed</span> on <span
| |
| - | class=SpellE>ice</span>, <span class=SpellE>add</span> in <span class=SpellE>order</span>:
| |
| - | <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- 25 uL 100
| |
| - | mg/mL tungsten (in 50 % <span class=SpellE>glycerol</span>) <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- 2 uL DNA (<span
| |
| - | class=GramE>0.5</span> mg / mL) <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- 25 uL <span
| |
| - | class=SpellE>CaCl</span><sub><span style='position:relative;top:5.0pt;
| |
| - | mso-text-raise:-5.0pt'>2</span></sub>, 2 M. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>- 10 uL <span
| |
| - | class=SpellE>Spermidine</span> <span class=SpellE>base</span>, <span
| |
| - | class=GramE>0.1</span> M. <o:p></o:p></span></p>
| |
| | | | |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>10. <span
| |
| - | class=SpellE>Incubate</span> on <span class=SpellE>ice</span> <span
| |
| - | class=SpellE>for</span> 10 min. <o:p></o:p></span></p>
| |
| - |
| |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>11. <span
| |
| - | class=SpellE>Spin</span> 1-2 <span class=SpellE>min</span> in <span
| |
| - | class=SpellE>microfuge</span>. <o:p></o:p></span></p>
| |
| - |
| |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>12. <span
| |
| - | class=SpellE>Remove</span> 25 uL of <span class=SpellE>the</span> <span
| |
| - | class=SpellE>supernatant</span>. Resuspend <span class=SpellE>the</span> rest <span
| |
| - | class=SpellE>by</span> <span class=SpellE>vortexing</span> <span class=SpellE>and</span>
| |
| - | a <span class=SpellE>brief</span> <span class=SpellE>sonication</span> (2-3 <span
| |
| - | class=SpellE>sec</span>) as <span class=SpellE>above</span>. <o:p></o:p></span></p>
| |
| - |
| |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>13. <span
| |
| - | class=SpellE>Apply</span> 8 uL <span class=SpellE>to</span> a <span
| |
| - | class=SpellE>filter</span> <span class=SpellE>holder</span>, <span
| |
| - | class=SpellE>attach</span> <span class=SpellE>to</span> <span class=SpellE>Helium</span>
| |
| - | <span class=SpellE>outlet</span>. <span class=SpellE>Place</span> a <span
| |
| - | class=SpellE>plate</span> in <span class=SpellE>the</span> <span class=SpellE>apparatus</span>
| |
| - | <span class=SpellE>and</span> <span class=SpellE>proceed</span> <span
| |
| - | class=SpellE>with</span> <span class=SpellE>bombardment</span>. (<span
| |
| - | class=SpellE><i>Parameters</i></span><i> <span class=SpellE>that</span> can be <span
| |
| - | class=SpellE>optimized</span> <span class=SpellE>include</span>: <span
| |
| - | class=SpellE>Helium</span> <span class=SpellE>pressure</span>, <span
| |
| - | class=SpellE>opening</span> time of <span class=SpellE>the</span> <span
| |
| - | class=SpellE>valve</span>, <span class=SpellE>pressure</span> in <span
| |
| - | class=SpellE>the</span> <span class=SpellE>chamber</span>, <span class=SpellE>distance</span>
| |
| - | <span class=SpellE>from</span> <span class=SpellE>the</span> <span
| |
| - | class=SpellE>sample</span> <span class=SpellE>holder</span> <span class=SpellE>to</span>
| |
| - | <span class=SpellE>the</span> <span class=SpellE>plate</span></i>). <o:p></o:p></span></p>
| |
| - |
| |
| - | <p class=Default><span style='font-size:11.5pt;color:windowtext'>14. <span
| |
| - | class=SpellE>Seal</span> <span class=SpellE>the</span> <span class=SpellE>plates</span>
| |
| - | <span class=SpellE>with</span> <span class=SpellE>Parafilm</span> (<span
| |
| - | class=SpellE>Micropore</span> <span class=SpellE>tape</span> <span
| |
| - | class=SpellE>for</span> minimal <span class=SpellE>medium</span>), <span
| |
| - | class=SpellE>and</span> <span class=SpellE>incubate</span> <span class=SpellE>under</span>
| |
| - | <span class=SpellE>appropriate</span> <span class=SpellE>conditions</span> <span
| |
| - | class=SpellE>for</span> <span class=SpellE>selection</span>. <span class=GramE>(</span><span
| |
| - | class=SpellE><i>If</i></span><i> <span class=SpellE>cells</span> <span
| |
| - | class=SpellE>were</span> <span class=SpellE>grown</span> <span class=SpellE>under</span>
| |
| - | <span class=SpellE>heterotrophic</span> <span class=SpellE>conditions</span> (<span
| |
| - | class=SpellE>acetate</span>, <span class=SpellE>dark</span>) <span
| |
| - | class=SpellE>and</span> <span class=SpellE>will</span> be <span class=SpellE>selected</span>
| |
| - | <span class=SpellE>for</span> <span class=SpellE>photoautotrophic</span> <span
| |
| - | class=SpellE>growth</span> (minimal, <span class=SpellE>light</span></i>)<i>,
| |
| - | put <span class=SpellE>the</span> <span class=SpellE>plates</span> in <span
| |
| - | class=SpellE>dim</span> <span class=SpellE>light</span> <span class=SpellE>for</span>
| |
| - | 16 – 24 <span class=SpellE>hours</span> <span class=SpellE>before</span> <span
| |
| - | class=SpellE>transferring</span> <span class=SpellE>to</span> <span
| |
| - | class=SpellE>light</span>. A ring of <span class=SpellE>colonies</span> <span
| |
| - | class=SpellE>will</span> <span class=SpellE>appear</span> <span class=SpellE>within</span>
| |
| - | 1-3 <span class=SpellE>weeks</span> <span class=SpellE>depending</span> on <span
| |
| - | class=SpellE>the</span> <span class=SpellE>selection</span> <span class=SpellE>applied</span>.<span
| |
| - | class=GramE>)</span> </i><o:p></o:p></span></p>
| |
| - |
| |
| - | <p class=Default><span class=SpellE><b><span style='font-size:11.5pt;
| |
| - | color:windowtext'>References</span></b></span><b><span style='font-size:11.5pt;
| |
| - | color:windowtext'>. </span></b><span style='font-size:11.5pt;color:windowtext'><o:p></o:p></span></p>
| |
| - |
| |
| - | <p class=Default style='text-indent:36.0pt'><span class=SpellE><span
| |
| - | style='font-size:11.5pt;color:windowtext'>Boynton</span></span><span
| |
| - | style='font-size:11.5pt;color:windowtext'> et al (1988) <span class=SpellE>Chloroplast</span>
| |
| - | <span class=SpellE>transformation</span> in Chlamydomonas <span class=SpellE>with</span>
| |
| - | <span class=SpellE>high</span> <span class=SpellE>velocity</span> <span
| |
| - | class=SpellE>microprojectiles</span>. <span class=SpellE>Science</span> 240:
| |
| - | 1534-1538. <o:p></o:p></span></p>
| |
| - |
| |
| - | <p class=MsoNormal><span class=SpellE><span style='font-size:11.5pt;line-height:
| |
| - | 115%'>Finer</span></span><span style='font-size:11.5pt;line-height:115%'> et
| |
| - | al. (1992) <span class=SpellE>Development</span> of <span class=SpellE>the</span>
| |
| - | <span class=SpellE>particle</span> <span class=SpellE>inflow</span> <span
| |
| - | class=SpellE>gun</span> <span class=SpellE>for</span> DNA <span class=SpellE>delivery</span>
| |
| - | <span class=SpellE>to</span> <span class=SpellE>plant</span> <span
| |
| - | class=SpellE>cells</span>. <span class=SpellE>Plant</span> <span class=SpellE>Cell</span>
| |
| - | <span class=SpellE>Reports</span> 11: 323-328.</span></p>
| |
| - | </div>
| |
| | | | |
| - | </body> | + | var popdiv=" "; |
| | + | for(var i =0;i<list.length;i++){ |
| | + | popdiv +="<DIV id=\"subcontent"+i+"\" class=\"pops\"></DIV>"; |
| | + | } |
| | + | $('.popingProblem').after(popdiv) |
| | + | for(var i=0 ;i<list.length;i++) |
| | + | dropdowncontent.init(("searchlink"+i), "right-bottom", 500, "mouseover"); |
| | + | //Call dropdowncontent.init("anchorID", "positionString", glideduration, "revealBehavior") at the end of the page: |
| | + | $('div').remove('.popingProblem',function(){ |
| | + | for(var i=0 ;i<list.length;i++) |
| | + | dropdowncontent.init(("searchlink"+i), "right-bottom", 500, "mouseover"); |
| | + | //Call dropdowncontent.init("anchorID", "positionString", glideduration, "revealBehavior") at the end of the page: |
| | + | }); |
| | + | |
| | + | </script> |
| | | | |
| | </html> | | </html> |
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