http://2010.igem.org/wiki/index.php?title=Team:Nevada/BY-2_(NT1)Transformation_Protocol&feed=atom&action=historyTeam:Nevada/BY-2 (NT1)Transformation Protocol - Revision history2024-03-28T10:14:21ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:Nevada/BY-2_(NT1)Transformation_Protocol&diff=193751&oldid=prevHilarya: /* BY-2 (NT1) Cell Transformation Protocol */2010-10-27T19:36:35Z<p><span class="autocomment">BY-2 (NT1) Cell Transformation Protocol</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>'''CARE OF BY-2 (NT1) CELL CULTURES'''</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>'''CARE OF BY-2 (NT1) CELL CULTURES'''</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>(<del class="diffchange diffchange-inline">adapted </del>from a letter by Dr. Michael Sullivan) </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>(<ins class="diffchange diffchange-inline">Adapted </ins>from a letter by Dr. Michael Sullivan) </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To readapt a culture on plates, simply transfer some of the cells back into liquid </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To readapt a culture on plates, simply transfer some of the cells back into liquid </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>media. We usually pipette the cell suspension up and down to break up any clumps. It </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>media. We usually pipette the cell suspension up and down to break up any clumps. It </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We grow our liquid cultures in 50 ml of media in 250 ml baffle flasks at 28 degrees C </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We grow our liquid cultures in 50 ml of media in 250 ml baffle flasks at 28 degrees C </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>with gentle shaking (150 rpm). Using a baffle flask does not seem to be a requirement<del class="diffchange diffchange-inline">, </del>many </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>with gentle shaking (150 rpm). Using a baffle flask does not seem to be a requirement<ins class="diffchange diffchange-inline">; </ins>many </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>people grow these cells in regular flasks with no problem. We <del class="diffchange diffchange-inline">subculture </del>them once a week </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>people grow these cells in regular flasks with no problem. We <ins class="diffchange diffchange-inline">subcultured </ins>them once a week </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>by transferring 5% of the culture to fresh media. We generally maintain two flasks </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>by transferring 5% of the culture to fresh media. We generally maintain two flasks </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>(in two separate shakers) of two separate subcultures (one subcultured Monday, one on Friday) </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>(in two separate shakers) of two separate subcultures (one subcultured Monday, one on Friday) </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>'''Day 2:''' </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>'''Day 2:''' </p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture which receives no bacteria. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture<ins class="diffchange diffchange-inline">, </ins>which receives no bacteria. </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when I'm finished. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when I'm finished. </div></td></tr>
</table>Hilaryahttp://2010.igem.org/wiki/index.php?title=Team:Nevada/BY-2_(NT1)Transformation_Protocol&diff=193350&oldid=prevEbersaba at 19:23, 27 October 20102010-10-27T19:23:57Z<p></p>
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</table>Ebersabahttp://2010.igem.org/wiki/index.php?title=Team:Nevada/BY-2_(NT1)Transformation_Protocol&diff=170320&oldid=prevBryson: /* BY-2 (NT1) Cell Transformation Protocol */2010-10-27T01:11:42Z<p><span class="autocomment">BY-2 (NT1) Cell Transformation Protocol</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>media. We usually pipette the cell suspension up and down to break up any clumps. It </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>media. We usually pipette the cell suspension up and down to break up any clumps. It </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>may be best to start out with a smaller culture volume when you first go back to liquid; </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>may be best to start out with a smaller culture volume when you first go back to liquid; </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>BY-2 seems to be a bit happier if it isn't seeded <del class="diffchange diffchange-inline">to </del>thinly. Allow the culture to grow </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>BY-2 seems to be a bit happier if it isn't seeded <ins class="diffchange diffchange-inline">too </ins>thinly. Allow the culture to grow </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>until it is the consistency of thin applesauce. At this point, you should be able to go to </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>until it is the consistency of thin applesauce. At this point, you should be able to go to </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>a 50 ml culture and start subculturing as described below. In our experience, wild-type </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>a 50 ml culture and start subculturing as described below. In our experience, wild-type </div></td></tr>
</table>Brysonhttp://2010.igem.org/wiki/index.php?title=Team:Nevada/BY-2_(NT1)Transformation_Protocol&diff=153410&oldid=prevHilarya at 03:02, 26 October 20102010-10-26T03:02:01Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Cell Transformation Protocol</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Cell Transformation Protocol</a></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium Transformations</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium Transformations</a></li></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><li><a href="https://2010.igem.org/Team:Nevada/Transgenic Plants" tabindex="3">Transgenic Plants: into the Wild</a></li></ins></div></td></tr>
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</table>Hilaryahttp://2010.igem.org/wiki/index.php?title=Team:Nevada/BY-2_(NT1)Transformation_Protocol&diff=152900&oldid=prevHilarya: /* BY-2 (NT1) Cell Transformation Protocol */2010-10-26T02:11:31Z<p><span class="autocomment">BY-2 (NT1) Cell Transformation Protocol</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>&nbsp;</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>&nbsp;</p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>== <del class="diffchange diffchange-inline">'''</del>BY-2 (NT1) Cell Transformation Protocol<del class="diffchange diffchange-inline">''' </del>==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>== BY-2 (NT1) Cell Transformation Protocol ==</div></td></tr>
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</table>Hilaryahttp://2010.igem.org/wiki/index.php?title=Team:Nevada/BY-2_(NT1)Transformation_Protocol&diff=152886&oldid=prevHilarya: /* BY-2 (NT1) Cell Transformation Protocol */2010-10-26T02:09:38Z<p><span class="autocomment">BY-2 (NT1) Cell Transformation Protocol</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/Results" tabindex="2">Results</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/Results" tabindex="2">Results</a></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Protocol</a></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT <ins class="diffchange diffchange-inline">Cell Transformation </ins>Protocol</a></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium</a></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li><a href="https://2010.igem.org/Team:Nevada/Agrobacterium Transformations" tabindex="3">Agrobacterium <ins class="diffchange diffchange-inline">Transformations</ins></a></li></div></td></tr>
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</table>Hilaryahttp://2010.igem.org/wiki/index.php?title=Team:Nevada/BY-2_(NT1)Transformation_Protocol&diff=152875&oldid=prevHilarya: /* BY-2 (NT1) Cell Transformation Protocol */2010-10-26T02:08:38Z<p><span class="autocomment">BY-2 (NT1) Cell Transformation Protocol</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>== BY-2 (NT1) Cell Transformation Protocol ==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>== <ins class="diffchange diffchange-inline">'''</ins>BY-2 (NT1) Cell Transformation Protocol<ins class="diffchange diffchange-inline">''' </ins>==</div></td></tr>
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</table>Hilaryahttp://2010.igem.org/wiki/index.php?title=Team:Nevada/BY-2_(NT1)Transformation_Protocol&diff=152728&oldid=prevHilarya: New page: {{Nevada_css}} 950px {{Nevada_topbar}} <div style="padding: 10px 10px 30px 10px;"> <p> </p> == BY-2 (NT1) Cell Transformation Prot...2010-10-26T01:50:59Z<p>New page: {{Nevada_css}} <a href="/File:NT_cell_transformations.png" title="File:NT cell transformations.png">950px</a> {{Nevada_topbar}} <div style="padding: 10px 10px 30px 10px;"> <p> </p> == BY-2 (NT1) Cell Transformation Prot...</p>
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<li><a href="https://2010.igem.org/Team:Nevada/BY-2 (NT1)Transformation Protocol" tabindex="1">NT Protocol</a></li><br />
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<p><span style="font-size:18px;font-weight:bold;text-decoration:underline">NT Cell Protocol</span></p><br />
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<p>'''CARE OF BY-2 (NT1) CELL CULTURES'''</p><br />
(adapted from a letter by Dr. Michael Sullivan) <br />
To readapt a culture on plates, simply transfer some of the cells back into liquid <br />
media. We usually pipette the cell suspension up and down to break up any clumps. It <br />
may be best to start out with a smaller culture volume when you first go back to liquid; <br />
BY-2 seems to be a bit happier if it isn't seeded to thinly. Allow the culture to grow <br />
until it is the consistency of thin applesauce. At this point, you should be able to go to <br />
a 50 ml culture and start subculturing as described below. In our experience, wild-type <br />
BY-2 readapts quickly to liquid to give a smooth culture; transformed lines tend to be more <br />
variable, with some producing smooth cultures, some producing clumpy ones, and some going <br />
back and forth between these two states. We've found that clumpy cultures do not interfere <br />
with our half-live measurements, although manipulating them can be a bit more difficult. <br />
<br />
We grow our liquid cultures in 50 ml of media in 250 ml baffle flasks at 28 degrees C <br />
with gentle shaking (150 rpm). Using a baffle flask does not seem to be a requirement, many <br />
people grow these cells in regular flasks with no problem. We subculture them once a week <br />
by transferring 5% of the culture to fresh media. We generally maintain two flasks <br />
(in two separate shakers) of two separate subcultures (one subcultured Monday, one on Friday) <br />
in case one of the cultures crashes. Also, you can maintain the culture on a plate. <br />
Note that, for liquid cultures of transformed lines, we usually use a smaller culture volume, <br />
e.g. 10 ml, for convenience. <br />
<br />
<br />
'''NT KC MEDIA - LIQUID OR PLATES<br />
NT LIQUID:''' <br />
(amounts are for 1L of media): <br />
750 ml H2O <br />
4.3 g MS salts (add slowly to liquid) <br />
30 g Sucrose <br />
50 ml 20X MES pH 5.7 <br />
10 ml B1 -inositol <br />
3 ml Miller's I <br />
10 ml 2,4-D, 10-4 M <br />
pH to 5.7 with 0.1 N KOH <br />
Bring volume to 1000ml <br />
Autoclave <br />
<br />
'''SOLID MEDIA:''' <br />
For plates only: Add to flasks 7 g/ L Phytagar before autoclaving <br />
Add kanamycin (100 mg/ml) <br />
Add carbenicillin (250 mg/ml) <br />
<br />
'''MEDIA COMPONENTS:''' <br />
Miller's I: 60 g KH2 PO4 per liter <br />
20 X MES: 10 g MES per liter, pH to 5.7 with 1N KOH <br />
B1 - Inositol: 0.1 g Thiamine, 10.0 g myo-inositol, H2O to final volume of 1 liter <br />
<br />
<br />
<br />
<p><span style="font-size:18px;font-weight:bold;text-decoration:underline">Transformation Protocol</span></p><br />
<p>'''BY-2 (NT1) Cell Transformation with Agrobactrium'''</p><br />
<p>'''Day 1:''' </p><br />
<p>1. Grow up 1 ml of the Agrobacterium overnight in LB + all selective drugs. This culture may be started from frozen glycerol cultures if necessary. </p><br />
<br />
<p>'''Day 2:''' </p><br />
<p>2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture which receives no bacteria. </p><br />
<br />
3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when I'm finished. <br />
<br />
4. Using a 10 ml pipette, the NT cells are pipetted in and out about 20 times. This helps to induce small lesions in the cells and increases the efficiency of the transformation. <br />
<br />
5. 75 ul of bacteria (dense growth) or 100 ul (moderate growth) are added to a petri dish containing 4 ml of NT cells (from step 4) and mixed thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA. <br />
<br />
6. Wrap plates with parafilm and incubate for 3 days at 28 degrees C. <br />
<br />
<p>'''Day 5:''' </p><br />
7. To each plate add 10 ml of NT liquid medium containing 500 (u)g/ml carbenicillin (NTC).<br />
<br />
8. Collect cells off of the plates into 50 ml conical tubes and fill with NTC. <br />
<br />
9. Centrifuge at 1K for 4 minutes at room temperature in a swinging bucket rotor. <br />
<br />
10. Aspirate off liquid using pipet capped with a sterile blue 1 ml top. Change tip for each sample.<br />
<br />
11. Resuspend in 50 ml NTC and repeat spin. <br />
<br />
12. Repeat step 10 and 11 - 1 or 2 times. Strains that are fairly sensitive to carb require fewer washes. <br />
<br />
13. Resuspend cells in approximately 5 ml NTC and plate 2.0 ml on 2 NTKC (kanamycin 100 ug/ml carbenicillin 500 ul/ml) plates. When there is no longer lots of fluid on the plates (leave the plates open in the hood a few minutes of necessary), wrap them in parafilm.<br />
<br />
14. Incubate at 28 degrees C. Transformants should be large enough to transfer to fresh NTKC plates in 3-4 weeks. <br />
<br />
'''Supplies for each transformation (Remember the controls):''' <br />
<br />
Day 1 Supplies: <br />
LB + appropriate drugs<br />
Agrobacterium containing plasmid for transformation <br />
<br />
Day 2 Supplies: <br />
1 ml Agrobacterium overnight culture <br />
4 ml BY-2 cells - 3 days post subculture <br />
4 ul acetosyringone (20 mM) found in the (-20) refrigerator freezer <br />
pipets and pipetman <br />
1 petri plate <br />
<br />
Day 5 Supplies: <br />
200 ml NTC liquid <br />
50 ml conical tube <br />
swinging bucket centrifuge at room temp aspiration setup with 5 ml pipet capped with 1 ml blue tip <br />
2 NTKC plates <br />
pipetmen and tips <br />
<br />
<br />
<br />
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<p>'''Left:''' NT Cell Culture after about 5 days. '''Right:''' NT Cell Transformation with RD29A (Cold, Drought, Salt) Inducible Promoter.</p><br />
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