July
From 2008.igem.org
(Difference between revisions)
(Replacing page with '{{Freiburg2008_Main| Content= <div style="font-size:18pt;"> <font face="Arial Rounded MT Bold" style="color:#010369">__july</font></div> <br> }}') |
m |
||
(27 intermediate revisions not shown) | |||
Line 2: | Line 2: | ||
Content= | Content= | ||
<div style="font-size:18pt;"> | <div style="font-size:18pt;"> | ||
- | <font face="Arial Rounded MT Bold" style="color:#010369"> | + | <font face="Arial Rounded MT Bold" style="color:#010369">_july</font></div> |
<br> | <br> | ||
+ | __NOTOC__ | ||
+ | <h3>Jul. 4th 2008</h3> | ||
+ | |||
+ | '''Assessment of Origami-Pool''' | ||
+ | |||
+ | We mixed all oligos, except for the modified, the left and the right borders and the remainders, together . | ||
+ | |||
+ | |||
+ | 1) '''Oligos modified with fluorophores''':<br> | ||
+ | <p> | ||
+ | 1. board:<br><br> | ||
+ | r-3t8f<br> | ||
+ | r-5t10e<br> | ||
+ | r-5t12e<br><br> | ||
+ | </p> | ||
+ | |||
+ | 2) '''Oligos modified with NIP''':<br> | ||
+ | <p> | ||
+ | 1. board:<br><br> | ||
+ | r-7t6f<br> | ||
+ | r-7t8f<br> | ||
+ | r-7t10f<br> | ||
+ | r-5t8f<br> | ||
+ | r-3t10e<br> | ||
+ | r-7t6e<br><br> | ||
+ | </p> | ||
+ | <p> | ||
+ | 3. board:<br><br> | ||
+ | r7t22e | ||
+ | </p> | ||
+ | |||
+ | |||
+ | '''3) Left border:''' 1.board, 1.line<br> | ||
+ | '''4) Right border:''' 3.board, 2.line<br> | ||
+ | '''5) Remainders:''' 3.board, 3.line<br> | ||
+ | <br><br> | ||
+ | <h3>Jul. 6th 2008</h3> | ||
+ | |||
+ | '''Assessment of cell cultures (DYT-Medium; ER 2738-cells)''' | ||
+ | |||
+ | 50ml DYT-Medium<br> | ||
+ | + 50 TET (25ng/ml -> 1:1000) <br> | ||
+ | + pick ER 2738-cells (from -80 degree freezer) <br> | ||
+ | <br> | ||
+ | -> shake at 37 degree over night | ||
+ | <br><br> | ||
+ | |||
+ | <h3>Jul. 7th 2008</h3> | ||
+ | '''1) Thinning down overnight culture to OD~0.1'''<br><br> | ||
+ | 1:10 -> 0.66 = OD 660<br><br> | ||
+ | 5000µl = 0.66<br> | ||
+ | x = 0.1 -> x = 758µl ~ 760µl in 50ml | ||
+ | <br><br> | ||
+ | |||
+ | We mixed 760µl cell culture with 50ml DYT and shaked both at 37°C<br> | ||
+ | <table cellspacing="0" cellpadding="3" width="100%" align="center" border="0"> | ||
+ | |||
+ | <tr> | ||
+ | <td> | ||
+ | <p align="center">t / min</p></td> | ||
+ | <td> | ||
+ | <p align="center">Assay</p></td> | ||
+ | <td> | ||
+ | <p align="center">OD (600)</p></td></tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p align="center">20</p></td> | ||
+ | <td> | ||
+ | <p align="center">1</p></td> | ||
+ | <td> | ||
+ | <p align="center">0,05</p></td></tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p align="center">60</p> | ||
+ | <p align="center">60</p></td> | ||
+ | <td> | ||
+ | <p align="center">1</p> | ||
+ | <p align="center">2</p></td> | ||
+ | <td> | ||
+ | <p align="center">0,28</p> | ||
+ | <p align="center">0,17</p></td></tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p align="center">75</p> | ||
+ | <p align="center">75</p></td> | ||
+ | <td> | ||
+ | <p align="center">1</p> | ||
+ | <p align="center">2</p></td> | ||
+ | <td> | ||
+ | <p align="center">0,37 *</p> | ||
+ | <p align="center">0,28</p></td></tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p align="center">95</p></td> | ||
+ | <td> | ||
+ | <p align="center">2</p></td> | ||
+ | <td> | ||
+ | <p align="center">0,39 *</p></td></tr></table> | ||
+ | <p>*inoculation of the culture</p> | ||
+ | <br> | ||
+ | '''2) Inoculation of a cell culture with M13mp18 phages'''<br> | ||
+ | <p>~ 50ml cellculture</p> | ||
+ | <p>+ | ||
+ | 5µl M13mp18 phages | ||
+ | (from -80°C)</p> | ||
+ | -> 4h shaking at 37°C | ||
+ | <br><br> | ||
+ | '''3) Phage precipitation''' | ||
+ | <p>1) centrifuging of the phage-solution at 5000 x g for 20 | ||
+ | min.</p> | ||
+ | <p>2) after decanting supernatant dessolve the pellet | ||
+ | in 7ml (1/7 of the supernatant volume) PEG/NaCl</p> | ||
+ | -> The precipitation occurs over night at 4°C | ||
+ | <br><br> | ||
+ | |||
+ | <h3>Jul. 8th 2008</h3> | ||
+ | |||
+ | '''1) Isolation of M13 phages''' | ||
+ | see Methods: Isolation of M13mp18 phages from the cellculture<br> | ||
+ | '''2) Measurement of the phage titer''' | ||
+ | -Photometer, Program: Spectrum Measurement<br> | ||
+ | -Absorption at 269nm<br> | ||
+ | -Measurement with Phage-Cuvette!<br> | ||
+ | -Thin down phages 1:30 or 1:40<br> | ||
+ | -Formula to calculate the Phagetiter:<br> | ||
+ | <br> | ||
+ | Phagetiter = (A269 * 6 * 10^16 * dilution factor) /(number of nucleotids in phage genom)<br> | ||
+ | |||
+ | ''Nucleotids in M13mp18-Phage genom = 7249 bp''<br> | ||
+ | <br> | ||
+ | Solution 1: (<u>0.36</u>*6*10^16*30) / 7249 = <u>8.9*10^13 phages/ml</u><br> | ||
+ | |||
+ | Solution 2: (<u>0.33</u>*6*10^16*30) / 7249 = <u>8.2*10^13 phages/ml</u><br><br> | ||
+ | ->the unit phages/ml have to be translated in µg DNA/ml because by purifying there should be at most 100µg DNA in a filter (Montage® PCR Centrifugal Filter Devices) | ||
+ | <br><br> | ||
+ | <p align="left"><strong><font size="2">3)Translation in µg DNA/ml</font></strong> </p> | ||
+ | Solution 1: 8.9*10^13 phages/ml ~ 8.9*10^13 particle/ml<br> | ||
+ | 8.9*10^13 particle = X1 | ||
+ | 6.022*10^23 particle = 1Mol<br> | ||
+ | <u> X1 = 1.5*10^-10Mol</u><br> | ||
+ | 1 Mol = 2.2*10^6g DNA | ||
+ | 1.5*10^-10Mol = X2<br> | ||
+ | <u> X2 = 3.3*10^-4g DNA -> 330µg DNA/ml</u><br> | ||
+ | Solution 2: | ||
+ | 6.022*10^23 particle = 1Mol | ||
+ | 8.2*10^13 particle = X1<br> | ||
+ | <u> X1 = 1.4*10^-10Mol</u><br> | ||
+ | 1 Mol = 2.2*10^6g DNA | ||
+ | 1.4*10^-10Mol = X2<br> | ||
+ | <u> X2 = 3.0*10^-4g DNA -> 300µg DNA/ml</u><br> | ||
+ | -> So we can use 250µl from each solution per round and filter<br><br> | ||
+ | '''4) Isolation of the phage DNA with QIAprep M13-Kit'''<br> | ||
+ | <p>- 1ml phages + 10µl MP-buffer, 2min. rest</p> | ||
+ | <p>- split in 4 filters inside an | ||
+ | Eppendorf-tube each 250µl</p> | ||
+ | <p>- centrifuge 8000g for 15sek.</p> | ||
+ | <p>- add 700µl MLP-buffer and centrifuge at 8000g for | ||
+ | 15sek., pour away buffer</p> | ||
+ | <p>- add 700µl MLP-buffer, 1min. rest, centrifuge at | ||
+ | 8000g for 15sek., pour away buffer</p> | ||
+ | <p>- add 700µl PE-buffer, centrifuge at 8000g for | ||
+ | 15sek., pour away buffer</p> | ||
+ | <p>- for drying, centrifuge again</p> | ||
+ | <p>Elution: put filters in fresh eppendorftubes and add | ||
+ | 100µl autoclaved Millipore-water on top of the membrane, inkubate for 10min.</p> | ||
+ | <p>- centrifuge out the water and the solute DNA (8000xg | ||
+ | for 30 sek.)</p> | ||
+ | <p>-> quantify DNA-concentration by drop...:</p> | ||
+ | <p>Solution 1:<u>448,4ng/ml</u></p> | ||
+ | <p>Solution 2:<u>423,4ng/ml</u></p> | ||
+ | |||
+ | |||
+ | <h3>Jul. 10th 2008</h3> | ||
+ | '''1) Origami'''(Simone+Michael)<br> | ||
+ | <br> | ||
+ | '''Origami without remainder'''<br> | ||
+ | 7.28µl------Oligos-delta-10(without the 10Oligos which could be marked)<br> | ||
+ | 1.18µl------M13pm18 phage DNA<br> | ||
+ | 0.4 µl------10 Oligos (Oligos which could be marked-> here we used the unmarked)<br> | ||
+ | 1.0 µl------MgAcetat(50x12,5mM Mg2+)<br> | ||
+ | -> add aqua-dest to 50µl<br> | ||
+ | <br> | ||
+ | '''Origami with remainder'''<br> | ||
+ | 7.28µl------Oligos-delta-10(without the 10Oligos which could be marked)<br> | ||
+ | 1.18µl------M13pm18 phage DNA<br> | ||
+ | 0.4 µl------10 Oligos (Oligos which could be marked-> here we used the unmarked)<br> | ||
+ | 0.4 µl------remainders<br> | ||
+ | 1.0 µl------MgAcetat(50x12,5mM Mg2+)<br> | ||
+ | -> add aqua-dest to 50µl<br> | ||
+ | <br> | ||
+ | The Origami were produced in a eppendorf Master cycler personal. Therefore they were heated up to 95°C for 7 minutes an then slowly cooled down (0,3°C/s) to 20°C.<br> | ||
+ | <br> | ||
+ | '''''Setting Master cycler'''''<br> | ||
+ | 1. T= 95,0° | ||
+ | 0:07:00<br> | ||
+ | 2. T= 90,0° | ||
+ | 0:01:00<br> | ||
+ | <div style="margin-left: 40px;"> -0,5° +0:00<br> | ||
+ | R=0,3°/s +0,0°/s<br> | ||
+ | </div> | ||
+ | 3. GOTO 2 | ||
+ | Rep 70<br> | ||
+ | 4. T= 55,0° | ||
+ | 0:01:00<br> | ||
+ | <div style="margin-left: 40px;">-0,5° +0:00<br> | ||
+ | R=0,3°/s +0,0°/s<br> | ||
+ | </div> | ||
+ | 5. GOTO 4 | ||
+ | Rep 70<br> | ||
+ | 6. HOLD | ||
+ | 20,0° Enter<br> | ||
+ | 7. end<br> | ||
+ | <br> | ||
+ | <br> | ||
+ | '''2) Purification of the DNA-origamis with Montage® PCR Centrifugal Filter Devices''' (Michael+Simone)<br> | ||
+ | To purify the DNA-Origamis of the unbound DNA-oligos we used | ||
+ | Montage® PCR Centrifugal Filter Devices (Millipore). | ||
+ | <br> | ||
+ | <br> | ||
+ | <ol> | ||
+ | <li>The Montage® PCR Centrifugal Filter Devices were | ||
+ | labeled and put in 1,5ml Eppendorf Tubes. The purple side has to be on | ||
+ | top.</li> | ||
+ | <li>To clean the Filter of remaining Glycerin: 450µl | ||
+ | TAE/MgAcetat (1x filtered) was put on top of the filter and for | ||
+ | 15minutes at 1000g (g=rcf) centrifuged. </li> | ||
+ | <li>The liquid in the Eppi was removed. </li> | ||
+ | <li>400µl TEA/MgAcetat + 45µl DNA-origami | ||
+ | were put on top of the filter and again centrifuged for 15minutes at | ||
+ | 1000g (g=rcf).</li> | ||
+ | <li>The liquid in the Eppi was removed. </li> | ||
+ | <li>To wash of all unbound DNA-oligos: 400µl | ||
+ | TEA/MgAcetat was put on top of the filter and for 15minutes at 1000g | ||
+ | (g=rcf) centrifuged. </li> | ||
+ | <li>To release the DNA-origamis of the filter put | ||
+ | 100µl TAE/MgAcetat on top of the filter and leave it at room | ||
+ | temperature at least for 2 minutes. (not to long, because the filter | ||
+ | shouldn´t run dry) </li> | ||
+ | <li>The Montage® PCR Centrifugal Filter Devices were | ||
+ | put upside down (the purple side has to be on bottom) in one of the | ||
+ | special Invert Spin tubes form Millipore and for 3 minutes at 1000g | ||
+ | centrifuged. | ||
+ | </li> | ||
+ | </ol> | ||
+ | ->The Origami were stored in the fridge(4°C).<br> | ||
+ | <br> | ||
+ | <h3>Jul. 11th 2008</h3><br> | ||
+ | <span style="font-weight: bold;">1) AFM measurement</span> (Michael+Simone+Daniel)<br> | ||
+ | To see if the Origami well formed and an atomic force microscope (AFM) was used.<br> | ||
+ | [[Image:Freiburg08_Bilder-vom-07-11-2008_1zu20_mit_und_ohne_rem.jpg|800 px]]<br> | ||
+ | ->all the Origami(with and without NIP) are formed well. So the remainders are not needed!<br> | ||
+ | <br> | ||
+ | <h3>Jul. 17th 2008</h3> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;">1) DNA-origamis in | ||
+ | different ratios</span><br> | ||
+ | This measurement was used to test if DNA-origami can be made at | ||
+ | a ratio of DNA-oligos to M13mp18 DNA smaller than 1:20.<br> | ||
+ | Three ratios were tested:<br> | ||
+ | Sample 1: DNA-oligos to M13mp18 DNA -> 1:20 (control)<br> | ||
+ | Sample 2: DNA-oligos to M13mp18 DNA -> 1:10 <br> | ||
+ | Sample 3: DNA-oligos to M13mp18 DNA -> 1: 5 <br> | ||
+ | <br> | ||
+ | We used different amount of M13mp18 DNA per sample!<br> | ||
+ | <ul> | ||
+ | <li>1:20; 4nM M13mp18 DNA</li> | ||
+ | <li>1:10; 8nM M13mp18 DNA</li> | ||
+ | <li>1:5; 12nM M13mp18 DNA<br> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <br style="font-weight: bold;"> | ||
+ | <span style="font-weight: bold;">2) Mastercycler</span><br> | ||
+ | Samples were putted in the Mastercycler as before. <br> | ||
+ | Program -> Origami 2<br> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;">3) purification of the | ||
+ | DNA-origamis</span><br> | ||
+ | To remove the unbound DNA-oligos the samples were washed as before | ||
+ | -> protocol 07-10-2008 <br> | ||
+ | Changes: <br> | ||
+ | To prove whether all of the DNA-origami were washed off the Millipore | ||
+ | we repeated step 5 and 6 of the protocol.<br> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;">4) Measurement on the AFM</span><br> | ||
+ | <br> | ||
+ | [[Image:TeamFreiGEM_Origamis2.jpg]] | ||
+ | <br> | ||
+ | <h3>Jul. 22nd 2008</h3> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;">1) Test Ringer-Solution</span><br> | ||
+ | With this essay, the stability of the DNA-Origami in | ||
+ | Ringer-Solution was tested.<br> | ||
+ | <br> | ||
+ | 4 probations were taken: <br> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;">1. Probation:</span> | ||
+ | we took M13mp18 DNA and DNA-oligos in a ratio of 1:20 (protocol from | ||
+ | 07-17-2008) and washed the origami in the Millipore-filter with | ||
+ | TEA/MgAcetat (control of 1:20)<br> | ||
+ | <span style="font-weight: bold;">2. Probation:</span> | ||
+ | we took M13mp18 DNA and DNA-oligos in a ratio of 1:20 (protocol from | ||
+ | 07-17-2008) and washed the origami in the Millipore-filter with the | ||
+ | Ringer-Solution (1:20 with Ringer)<br> | ||
+ | <span style="font-weight: bold;">3. Probation:</span> | ||
+ | we took M13mp18 DNA and DNA-oligos in a ratio of 1:5 (protocol from | ||
+ | 07-17-2008) and washed the origami in the Millipore-filter with | ||
+ | TEA/MgAcetat (control of 1:5)<br> | ||
+ | <span style="font-weight: bold;">4. Probation:</span> | ||
+ | we took M13mp18 DNA and DNA-oligos in a ratio of 1:5 (protocol from | ||
+ | 07-17-2008) and washed the origami in the Millipore-filter with the | ||
+ | Ringer-Solution (1:5 with Ringer)<br> | ||
+ | <br> | ||
+ | The origami were made as before -> protocol from 07-17-2008<br> | ||
+ | The origamis were purified as before. Except for the probations we | ||
+ | wanted to test the Ringer-Solution we used the Ringer-Solution instead | ||
+ | of TEA/MgAcetat -> protocol from 07-10-2008<br> | ||
+ | <br> | ||
+ | If the origamis are also stable in the Ringer-Solution we should see | ||
+ | well formed DNA-origami in the AFM.<br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <h3>Jul. 23rd 2008</h3> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;">1) Measurement on the AFM</span><br> | ||
+ | [[image:Freiburg08_Bilder-vom-07-23-2008_1zu20_kontrolle_und_Ringer.jpg| 800 px]]<br> | ||
+ | [[image:Freiburg08_Bilder-vom-07-23-2008_1zu5_kontrolle_und_Ringer.jpg|800 px]]<br><br> | ||
+ | We couldn’t see any DNA-origami!!! <br> | ||
+ | -> may be the origami are not stable in the Ringer-Solution <br> | ||
+ | or<br> | ||
+ | -> The NaCl in the Ringer-Solution disturbed the binding between | ||
+ | the glimmer-surface and the origami. To test this, the concentrations | ||
+ | of the four probations were measured | ||
+ | ……… in the Nano-drop. If the | ||
+ | concentrations of the probations with Ringer-Solution is comparable to | ||
+ | the concentrations of the controls, than we should purify the | ||
+ | DNA-origami again, using TAE/MgAcetat as buffer.<br> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;">2) Measurement on the | ||
+ | Nano-Trop</span><br> | ||
+ | <br> | ||
+ | <table style="text-align: left; width: 100px;" border="1" | ||
+ | cellpadding="2" cellspacing="2"> | ||
+ | <tr> | ||
+ | <td>Sample</td> | ||
+ | <td>ng/µl</td> | ||
+ | <td>A260</td> | ||
+ | <td>A280</td> | ||
+ | <td>260/280</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1:20 with Ringer</td> | ||
+ | <td>4.19</td> | ||
+ | <td>0.105</td> | ||
+ | <td>0.075</td> | ||
+ | <td>1.40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1:20 with Ringer</td> | ||
+ | <td>4.46</td> | ||
+ | <td>0.111</td> | ||
+ | <td>0.078</td> | ||
+ | <td>1.43</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1:5 with Ringer</td> | ||
+ | <td>18.33</td> | ||
+ | <td>0.458</td> | ||
+ | <td>0.277</td> | ||
+ | <td>1.65</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1:5 with Ringer</td> | ||
+ | <td>15.96</td> | ||
+ | <td>0.399</td> | ||
+ | <td>0.248</td> | ||
+ | <td>1.61</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>control of 1:20</td> | ||
+ | <td>5.94</td> | ||
+ | <td>0.148</td> | ||
+ | <td>0.097</td> | ||
+ | <td>1.53</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>control of 1:5</td> | ||
+ | <td>14.16</td> | ||
+ | <td>0.354</td> | ||
+ | <td>0.200</td> | ||
+ | <td>1.77</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | Probation 2 showed nearly the same concentration as probation 1.<br> | ||
+ | Probation 4 showed nearly the same concentration as probation 3.<br> | ||
+ | -> There might by well formed origami in the Ringer-Solution. To | ||
+ | test this further, the probations with Ringer-Solution were purified | ||
+ | and buffered with TAE/MgAcetat.<br> | ||
+ | <br style="font-weight: bold;"> | ||
+ | <span style="font-weight: bold;">3) buffer the DNA-origami | ||
+ | from Ringer-Solution in TAE/MgAcetat</span><br> | ||
+ | The DNA-origamis in Ringer-Solution were purified as before (-> | ||
+ | purification protocol 07-10-2008)<br> | ||
+ | Changes of the protocol: <br> | ||
+ | -> Instead of 45µl of Origami we putted | ||
+ | 100µl of our DNA-origami in Ringer-Solution (07-22-2008, | ||
+ | probation 2 and 3) on the Filter (Step4).<br> | ||
+ | <br style="font-weight: bold;"> | ||
+ | <span style="font-weight: bold;">4) Order new | ||
+ | Montage® PCR Centrifugal Filter Devices</span><br> | ||
+ | We ordered new Montage® PCR Centrifugal Filter Devices at | ||
+ | Millipore online.<br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <h3>Jul. 24th 2008</h3> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;">1) DNA-Origamis with NIP | ||
+ | and fluorophor</span><br> | ||
+ | For the measurement at the fluorescence microscope we prepared some | ||
+ | DNA-oligos with NIPs and fluorophor.<br> | ||
+ | First we wanted to test if the DNA-origami with NIPs bind to the cells. | ||
+ | For this we prepared DNA-origami coupled with NIPs and fluorophor and | ||
+ | DNA-origami coupled with fluorophor but without NIPs (negative | ||
+ | control). Ratio M13mp18 DNA to Oligos 1:5.<br> | ||
+ | As fluorophor we took Alexa488 (λexc= 488 nm, | ||
+ | λem= 515 nm) and we used all of the 7NIPs.<br> | ||
+ | <br> | ||
+ | Second we wanted to test if the DNA-Origami with NIPs induces cellular | ||
+ | signalling by measuring the calcium influx. For this we prepared | ||
+ | DNA-origami with NIPs and DNA-origami without NIPs (negative control). | ||
+ | Ratio M13mp18 DNA to Oligos 1:5.<br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <h3>Jul. 29th 2008</h3> | ||
+ | <br> | ||
+ | '''1) Measurement at the AFM<br>''' | ||
+ | We measured the origami which we produced in Ringer-Solution. Because | ||
+ | we didn´t see any origami in the last measurement (07-23-08) | ||
+ | we buffered them in TAE/MgAc and measured them again.<br> | ||
+ | BILD<br> | ||
+ | We couldn’t see any origami only some greasy spots which were | ||
+ | smaller (50-70 nm) then the right ones (100 nm).<br> | ||
+ | <br> | ||
+ | '''Improvements:<br>''' | ||
+ | 1.) We could put more Mg into the Ringer-Solution to boost the | ||
+ | DNA-backbone.<br> | ||
+ | 2.) If the T-cells tolerate the TAE/MgAc in small concentrations we | ||
+ | could use this buffer to produce the origami.<br> | ||
+ | <br> | ||
+ | '''2) Purification of the DNA-origamis <br>''' | ||
+ | We purified the origami with fluorophor, with fluorophor and NIPs, just | ||
+ | with NIPs and without NIPs or fluorophor (Control) from 07-24-2008. <br> | ||
+ | -> Protocol from 07-10-2008<br> | ||
+ | Changes: instead of 45 µl we used 65 µl of the | ||
+ | DNA-origami.<br> | ||
+ | <br><br> | ||
+ | |||
+ | |||
+ | <h3>Jul. 31st 2008</h3> | ||
+ | <br> | ||
+ | |||
+ | <p align="left"><font size="2"><strong> T-cell staining</strong></font> | ||
+ | <br> | ||
+ | 1) The T-cells of 2 falcontubes were spun down at 1200rpm for 5 min. The supernatant was thrown away.<br> | ||
+ | 2) The pellets were resuspended in 25ml RPMI and again spun down at 1200rpm for 5 min. The supernatant was thrown away.<br> | ||
+ | 3) The pellet was resuspendet in 11ml RPMI (with 1% FCS). For two new cultures we put in both petri dish 5ml of the cell suspension.<br> The remaining 1ml was spun down (as before). The supernatant was thrown away.<br> | ||
+ | 4) 1ml RPMI with 1%FCS, 0,5µl Fura2AM and 0,625µl Pluronic were put in an 1,5ml Eppendorf-Tube and mixed on the vortexer.<br> With this mixture we resuspended the pellet in the falcon tube(cells). <br> | ||
+ | |||
+ | |||
+ | -> shaking for 30minutes at room temperature.<br> | ||
+ | <br> | ||
+ | <font size="2"><strong>2) Wash the cells and buffer it in the | ||
+ | Ringer-Solution</strong></font></p> | ||
+ | <p>For the measurement we have to buffer the cells in Ringer-Solution, because | ||
+ | the phenol red in the RPMI could disturb the measurement. Therefore we washed | ||
+ | the cells with 10ml of Ringer-Solution.</p> | ||
+ | <p>10ml Ringer-Solution was put in the falcon tube with the cells and | ||
+ | centrifuged at 1200rpm for 4minutes. (Normally the cells should be centrifuged | ||
+ | for 5minutes at 1200rpm.). The supernatant was thrown away and the pellet was | ||
+ | resuspended in 100µl Ringer-Solution. 10µl of the cells were put in each well of | ||
+ | the slide.</p><i> | ||
+ | <p>Changes: Normally the cells should be washed twice, but by throwing away the | ||
+ | supernatant we lost lots of cells, so we didn´t wash it a second time. Also the | ||
+ | cell pellet should be resuspended in 200µl instead of 100µl.</p></i> | ||
+ | |||
+ | <font size="2"><strong>3) Calcium measurement on the | ||
+ | microscope</strong></font><br> | ||
+ | Activators:<br> | ||
+ | <p>1) 1µl NIP<sub>15</sub> – BSA in 100µl Ringer-Solution.</p> | ||
+ | <p>2) 1µl NaVO<sub>3</sub> in 100µl Ringer-Solution. NaVO<sub>3</sub> inhibits | ||
+ | the Phosphotase, which dephosphorylate some cell structures this leads to | ||
+ | calcium influx (positive control).</p> | ||
+ | <p>3) 1µl Anti-CD28-antibody in 100µl Ringer-Solution.</p> | ||
+ | <p>Measurement:</p> | ||
+ | <p>The calcium influx was measured at 340nm and 380nm. The quotient of both | ||
+ | (340/380) changes when the cells absorb the calcium | ||
+ | from the outside. The calcium is absorbed when the TCR gets activated.</p> | ||
+ | <p>When Fura2 is bound to calcium it shows more signal at 380nm and less signal | ||
+ | at 340nm.</p> | ||
+ | <p>1. + 1µl NIP<sub>15</sub> – BSA in one well -> nothing happened</p> | ||
+ | <p>2. +10µl NIP<sub>15</sub> – BSA in one well -> nothing happened</p> | ||
+ | <p>3. +10µl Pervanadat in one well -> nothing happened</p> | ||
+ | <p>-> We guess that the cells were not labelled properly, because normally | ||
+ | the FURA 2 stained cells bleach over time. But our cells didn´t bleach at | ||
+ | all.</p> | ||
+ | <p> </p> | ||
+ | <p>Because of this we stained the cells again. We put 10µl of Fura2AM on top of | ||
+ | the unused cells in the wells and incubated it for 30minutes at room | ||
+ | temperature. We didn´t wash the cells but put directly the activators on it. | ||
+ | </p> | ||
+ | <p>Measurement: </p> | ||
+ | <p>1. 10µl Pervanadat -> we could see a change of | ||
+ | fluorescence which was most likely caused of the calcium influx, because the activity at 380nm increased while the signal at 340nm decreased.</p> | ||
+ | |||
+ | <p align="left"><font size="2"><strong>4) Measure the binding of DNA-Origami to the cells</strong></font></p> | ||
+ | <p>We wanted to test if the DNA-origamis with 7NIPs and flourophor and the | ||
+ | DNA-origami without NIPs but with fluorophor (negative control) from 07-24-2008 | ||
+ | bind to the T-cells.</p> | ||
+ | <p>The cells were measured on an inverse fluorescent microscope (excitation: | ||
+ | 488nm, measured at 500-550nm).</p> | ||
+ | <p>We also used the FURA stained cells because we didn´t have any others, but | ||
+ | better is to use unstained cells.</p> | ||
+ | <p>Test solutions (in 1,5ml eppendorf tube): </p> | ||
+ | <p>1. 10µl of stained cells (-> 1) T-cell staining)</p> | ||
+ | |||
+ | <p>+ 10µl of the DNA-origamis with NIPs and flourophor(Alexa | ||
+ | 488)</p> | ||
+ | <p>2. 10µl of stained cells (-> 1) T-cell staining)</p> | ||
+ | |||
+ | <p>+ 10µl of the DNA-origamis with flourophor (Alexa 488), but without | ||
+ | NIPs</p> | ||
+ | <p>-> Both solutions were mix in a 1,5ml eppendorf tube and then out on the | ||
+ | wells.</p> | ||
+ | <p>Procedure: </p> | ||
+ | <p>To see if the cells are fine we put 10µl of the stained cells directly in one | ||
+ | well. At the same time we put the test-solution 1 on another well. We measured | ||
+ | the cells. When we looked at these two probations we saw that not the whole well | ||
+ | was covered with the solutions so we decided to put more volume in it. Therefore | ||
+ | we put directly 10µl Ringer-Solution in the well with the stained cells only and | ||
+ | 10µl of the DNA-Origamis with NIPs and fluorophor in the corresponding well. | ||
+ | Also we put 10µl of DNA-origamis with flourophor, but without NIPs in the | ||
+ | corresponding eppendorf tube, mixed it and also put it on a well.</p> | ||
+ | <p>Outcomes:</p> | ||
+ | <p>In both probations (DNA-origamis with and without NIPS) bound to the T-cells | ||
+ | surface. In the beginning it seemed that the DNA-Origamis without NIP do not | ||
+ | show as much signal as the one with NIPs, but this could basically be caused of | ||
+ | to thinks:</p> | ||
+ | <p>1. They weren´t illuminated as long as the T-cells with NIP coupled | ||
+ | DNA-Origami.</p> | ||
+ | <p>2. The Cells with the DNA-origamis with flourophor, but without NIPs were | ||
+ | much better mixed than the other ones, because we could mix the whole test | ||
+ | solution before putting it on the well.</p> | ||
+ | <br> | ||
+ | <p>improvements in general: </p> | ||
+ | <p>1. We should always bring some unstained cells, to have some comparison. For | ||
+ | example to see if the stained cells are probably stained or if the fluorescence | ||
+ | we see comes from the cells autofluorescens</p> | ||
+ | <p>2. We should use 20µl of the cell-culture (or 10µl of cells with 10µl of | ||
+ | Ringer-Solution) so that the whole well is covered with liquid. </p> | ||
+ | |||
}} | }} |
Latest revision as of 02:40, 30 October 2008