July
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<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__ | __NOTOC__ | ||
<h3>Jul. 4th 2008</h3> | <h3>Jul. 4th 2008</h3> | ||
- | ''' | + | '''Assessment of Origami-Pool''' |
- | We mixed all oligos | + | We mixed all oligos, except for the modified, the left and the right borders and the remainders, together . |
- | 1) Oligos modified with | + | 1) '''Oligos modified with fluorophores''':<br> |
<p> | <p> | ||
1. board:<br><br> | 1. board:<br><br> | ||
Line 20: | Line 20: | ||
</p> | </p> | ||
- | 2) Oligos modified with NIP:<br> | + | 2) '''Oligos modified with NIP''':<br> |
<p> | <p> | ||
1. board:<br><br> | 1. board:<br><br> | ||
Line 30: | Line 30: | ||
r-7t6e<br><br> | r-7t6e<br><br> | ||
</p> | </p> | ||
- | |||
<p> | <p> | ||
3. board:<br><br> | 3. board:<br><br> | ||
Line 37: | Line 36: | ||
- | 3) Left border: 1.board, 1.line<br> | + | '''3) Left border:''' 1.board, 1.line<br> |
- | 4) Right border: 3.board, 2.line<br> | + | '''4) Right border:''' 3.board, 2.line<br> |
- | 5) Remainders: 3.board, 3.line<br> | + | '''5) Remainders:''' 3.board, 3.line<br> |
<br><br> | <br><br> | ||
<h3>Jul. 6th 2008</h3> | <h3>Jul. 6th 2008</h3> | ||
- | ''' | + | '''Assessment of cell cultures (DYT-Medium; ER 2738-cells)''' |
50ml DYT-Medium<br> | 50ml DYT-Medium<br> | ||
Line 53: | Line 52: | ||
<h3>Jul. 7th 2008</h3> | <h3>Jul. 7th 2008</h3> | ||
- | '''1) | + | '''1) Thinning down overnight culture to OD~0.1'''<br><br> |
1:10 -> 0.66 = OD 660<br><br> | 1:10 -> 0.66 = OD 660<br><br> | ||
5000µl = 0.66<br> | 5000µl = 0.66<br> | ||
Line 59: | Line 58: | ||
<br><br> | <br><br> | ||
- | We mixed 760µl | + | 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"> | <table cellspacing="0" cellpadding="3" width="100%" align="center" border="0"> | ||
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<p>*inoculation of the culture</p> | <p>*inoculation of the culture</p> | ||
<br> | <br> | ||
- | '''2) | + | '''2) Inoculation of a cell culture with M13mp18 phages'''<br> |
<p>~ 50ml cellculture</p> | <p>~ 50ml cellculture</p> | ||
<p>+ | <p>+ | ||
Line 117: | Line 116: | ||
<p>2) after decanting supernatant dessolve the pellet | <p>2) after decanting supernatant dessolve the pellet | ||
in 7ml (1/7 of the supernatant volume) PEG/NaCl</p> | in 7ml (1/7 of the supernatant volume) PEG/NaCl</p> | ||
- | -> The precipitation occurs | + | -> The precipitation occurs over night at 4°C |
<br><br> | <br><br> | ||
- | <h3>Jul. 8th 2008</h3> | + | |
+ | <h3>Jul. 8th 2008</h3> | ||
+ | |||
'''1) Isolation of M13 phages''' | '''1) Isolation of M13 phages''' | ||
- | + | see Methods: Isolation of M13mp18 phages from the cellculture<br> | |
- | phages from the cellculture | + | |
'''2) Measurement of the phage titer''' | '''2) Measurement of the phage titer''' | ||
-Photometer, Program: Spectrum Measurement<br> | -Photometer, Program: Spectrum Measurement<br> | ||
-Absorption at 269nm<br> | -Absorption at 269nm<br> | ||
- | -Measurement with | + | -Measurement with Phage-Cuvette!<br> |
-Thin down phages 1:30 or 1:40<br> | -Thin down phages 1:30 or 1:40<br> | ||
-Formula to calculate the Phagetiter:<br> | -Formula to calculate the Phagetiter:<br> | ||
Line 154: | Line 154: | ||
1.4*10^-10Mol = X2<br> | 1.4*10^-10Mol = X2<br> | ||
<u> X2 = 3.0*10^-4g DNA -> 300µg DNA/ml</u><br> | <u> X2 = 3.0*10^-4g DNA -> 300µg DNA/ml</u><br> | ||
- | -> So we can use | + | -> 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> | |
- | phage DNA with QIAprep M13-Kit< | + | |
<p>- 1ml phages + 10µl MP-buffer, 2min. rest</p> | <p>- 1ml phages + 10µl MP-buffer, 2min. rest</p> | ||
<p>- split in 4 filters inside an | <p>- split in 4 filters inside an | ||
- | + | Eppendorf-tube each 250µl</p> | |
- | <p>- centrifuge | + | <p>- centrifuge 8000g for 15sek.</p> |
- | <p>- add 700µl MLP-buffer and centrifuge | + | <p>- add 700µl MLP-buffer and centrifuge at 8000g for |
15sek., pour away buffer</p> | 15sek., pour away buffer</p> | ||
- | <p>- add 700µl MLP-buffer, 1min. rest, centrifuge | + | <p>- add 700µl MLP-buffer, 1min. rest, centrifuge at |
- | + | 8000g for 15sek., pour away buffer</p> | |
- | <p>- add 700µl PE-buffer, centrifuge | + | <p>- add 700µl PE-buffer, centrifuge at 8000g for |
15sek., pour away buffer</p> | 15sek., pour away buffer</p> | ||
- | <p>- for drying centrifuge again</p> | + | <p>- for drying, centrifuge again</p> |
<p>Elution: put filters in fresh eppendorftubes and add | <p>Elution: put filters in fresh eppendorftubes and add | ||
100µl autoclaved Millipore-water on top of the membrane, inkubate for 10min.</p> | 100µl autoclaved Millipore-water on top of the membrane, inkubate for 10min.</p> | ||
Line 173: | Line 172: | ||
for 30 sek.)</p> | for 30 sek.)</p> | ||
<p>-> quantify DNA-concentration by drop...:</p> | <p>-> quantify DNA-concentration by drop...:</p> | ||
- | <p>Solution 1: | + | <p>Solution 1:<u>448,4ng/ml</u></p> |
- | <p>Solution 2: | + | <p>Solution 2:<u>423,4ng/ml</u></p> |
- | + | <h3>Jul. 10th 2008</h3> | |
- | <h3>07- | + | '''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> | <br> | ||
<span style="font-weight: bold;">1) DNA-origamis in | <span style="font-weight: bold;">1) DNA-origamis in | ||
different ratios</span><br> | different ratios</span><br> | ||
- | This measurement was used to test if DNA-origami | + | 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> | Three ratios were tested:<br> | ||
Sample 1: DNA-oligos to M13mp18 DNA -> 1:20 (control)<br> | Sample 1: DNA-oligos to M13mp18 DNA -> 1:20 (control)<br> | ||
Line 189: | Line 267: | ||
Sample 3: DNA-oligos to M13mp18 DNA -> 1: 5 <br> | Sample 3: DNA-oligos to M13mp18 DNA -> 1: 5 <br> | ||
<br> | <br> | ||
- | We used different M13mp18 DNA per sample!<br> | + | We used different amount of M13mp18 DNA per sample!<br> |
<ul> | <ul> | ||
- | <li> | + | <li>1:20; 4nM M13mp18 DNA</li> |
<li>1:10; 8nM M13mp18 DNA</li> | <li>1:10; 8nM M13mp18 DNA</li> | ||
<li>1:5; 12nM M13mp18 DNA<br> | <li>1:5; 12nM M13mp18 DNA<br> | ||
Line 209: | Line 287: | ||
we repeated step 5 and 6 of the protocol.<br> | we repeated step 5 and 6 of the protocol.<br> | ||
<br> | <br> | ||
+ | <span style="font-weight: bold;">4) Measurement on the AFM</span><br> | ||
+ | <br> | ||
+ | [[Image:TeamFreiGEM_Origamis2.jpg]] | ||
<br> | <br> | ||
- | <h3> | + | <h3>Jul. 22nd 2008</h3> |
<br> | <br> | ||
<span style="font-weight: bold;">1) Test Ringer-Solution</span><br> | <span style="font-weight: bold;">1) Test Ringer-Solution</span><br> | ||
- | With this essay | + | With this essay, the stability of the DNA-Origami in |
- | + | Ringer-Solution was tested.<br> | |
<br> | <br> | ||
- | + | 4 probations were taken: <br> | |
<br> | <br> | ||
<span style="font-weight: bold;">1. Probation:</span> | <span style="font-weight: bold;">1. Probation:</span> | ||
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<br> | <br> | ||
<br> | <br> | ||
- | <h3> | + | <h3>Jul. 23rd 2008</h3> |
<br> | <br> | ||
<span style="font-weight: bold;">1) Measurement on the AFM</span><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]]<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]]<br><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> | We couldn’t see any DNA-origami!!! <br> | ||
-> may be the origami are not stable in the Ringer-Solution <br> | -> may be the origami are not stable in the Ringer-Solution <br> | ||
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concentrations of the probations with Ringer-Solution is comparable to | concentrations of the probations with Ringer-Solution is comparable to | ||
the concentrations of the controls, than we should purify the | the concentrations of the controls, than we should purify the | ||
- | DNA-origami again | + | DNA-origami again, using TAE/MgAcetat as buffer.<br> |
<br> | <br> | ||
<span style="font-weight: bold;">2) Measurement on the | <span style="font-weight: bold;">2) Measurement on the | ||
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<br> | <br> | ||
<br> | <br> | ||
- | <h3> | + | <h3>Jul. 24th 2008</h3> |
<br> | <br> | ||
<span style="font-weight: bold;">1) DNA-Origamis with NIP | <span style="font-weight: bold;">1) DNA-Origamis with NIP | ||
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<br> | <br> | ||
<br> | <br> | ||
- | <h3> | + | <h3>Jul. 29th 2008</h3> |
<br> | <br> | ||
'''1) Measurement at the AFM<br>''' | '''1) Measurement at the AFM<br>''' | ||
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- | <h3> | + | <h3>Jul. 31st 2008</h3> |
<br> | <br> | ||
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much better mixed than the other ones, because we could mix the whole test | much better mixed than the other ones, because we could mix the whole test | ||
solution before putting it on the well.</p> | solution before putting it on the well.</p> | ||
- | < | + | <br> |
- | <p>improvements in | + | <p>improvements in general: </p> |
<p>1. We should always bring some unstained cells, to have some comparison. For | <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 | example to see if the stained cells are probably stained or if the fluorescence |
Latest revision as of 02:40, 30 October 2008