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Team:Freiburg/Project/toolkit
From 2013.igem.org
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Therefore you have to order the following plasmids from the <a id="link" | Therefore you have to order the following plasmids from the <a id="link" | ||
| - | href="http://parts.igem.org/Main_Page"> iGEM parts registry </a> | + | href="http://parts.igem.org/Main_Page"> iGEM parts registry </a>. After receiving our plasmids, you will have to clone your target sequence into our crRNA plasmid (protocol see below). |
</p> | </p> | ||
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</table> | </table> | ||
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<p> | <p> | ||
Note: <br> | Note: <br> | ||
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<ol> | <ol> | ||
| - | <li> Oligo annealing: Anneal forward and reverse oligo to get the | + | <li> <b>Oligo annealing:</b> Anneal forward and reverse oligo to get the |
desired crRNA. Therefore mix 10 µl of 100 µM forward Oligo, 10 µl of 100 µM reverse Oligo and 80 | desired crRNA. Therefore mix 10 µl of 100 µM forward Oligo, 10 µl of 100 µM reverse Oligo and 80 | ||
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solution cool down.</li> | solution cool down.</li> | ||
| - | <li> Digest plasmid BBa_K1150034 with Bbs1: The restriction enzyme | + | <li> <b>Digest plasmid BBa_K1150034 with Bbs1:</b> The restriction enzyme |
Bbs1 should always be stored at -80° C. Mix about 500 ng of BBa_K1150034 with 1 µl of Bbs1, | Bbs1 should always be stored at -80° C. Mix about 500 ng of BBa_K1150034 with 1 µl of Bbs1, | ||
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</li> | </li> | ||
| - | <li> Ligate crRNAs (step 1) into Bbs1 cut backbone: The insert | + | <li> <b>Ligate crRNAs (step 1) into Bbs1 cut backbone:</b> The insert |
(crRNAs) should be ligated into the backbone in 3 molar insert excess. Therefore use this | (crRNAs) should be ligated into the backbone in 3 molar insert excess. Therefore use this | ||
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minutes at room temperature.</li> | minutes at room temperature.</li> | ||
| - | <li> Transform 3-5 µl of the mix following standard protocol. Pick | + | <li> <b>Transform</b> 3-5 µl of the mix following standard protocol. Pick |
clones, miniprep the plasmids and sequence it with pSB1C3 reverse sequencing primer (sequence: | clones, miniprep the plasmids and sequence it with pSB1C3 reverse sequencing primer (sequence: | ||
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</p> | </p> | ||
<ol> | <ol> | ||
| - | <li> Digest first crRNA plasmid with XXX and XXX. This is your | + | <li> <b>Digest first crRNA plasmid</b> with XXX and XXX. This is your |
backbone. Therefore mix about 500 ng Backbone with 1 µl Enzyme 1 and 1 µl Enzyme 2, add an | backbone. Therefore mix about 500 ng Backbone with 1 µl Enzyme 1 and 1 µl Enzyme 2, add an | ||
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for 2 hours. </li> | for 2 hours. </li> | ||
| - | <li> Digest second crRNA plasmid with XXX and XXX. This is your | + | <li> <b>Digest second crRNA plasmid</b> with XXX and XXX. This is your |
insert (procedure see above).</li> | insert (procedure see above).</li> | ||
| - | <li> Ligate the insert in 3 molar excess into the backbone | + | <li> <b>Ligate</b> the insert in 3 molar excess into the backbone |
(formular see paragraph above). | (formular see paragraph above). | ||
| - | <li> Transform 3-5 µl of the mix following standard protocol. Pick | + | <li> <b>Transform</b> 3-5 µl of the mix following standard protocol. Pick |
clones, miniprep the plasmids and sequence it with pSB1C3 forward sequencing primer (sequence: | clones, miniprep the plasmids and sequence it with pSB1C3 forward sequencing primer (sequence: | ||
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</p> | </p> | ||
<ol> | <ol> | ||
| - | <li> Transfect BBa_K1150020 with all desired crRNA plasmids | + | <li> <b>Transfect</b> BBa_K1150020 with all desired crRNA plasmids |
(seperate crRNA plasmid and/or multiple crRNAs plasmid) </li> | (seperate crRNA plasmid and/or multiple crRNAs plasmid) </li> | ||
| - | <li> Non-effector control: Transfect the appropriate crRNA - | + | <li> <b>Non-effector control:</b> Transfect the appropriate crRNA - |
plasmid togehter with BBa_K1150017 that has no effector.</li> | plasmid togehter with BBa_K1150017 that has no effector.</li> | ||
| - | <li> Off target control: Transfect BBa_K1150020 without any crRNA | + | <li> <b>Off target control:</b> Transfect BBa_K1150020 without any crRNA |
plasmid.</li> | plasmid.</li> | ||
Revision as of 16:08, 29 September 2013
The uniCAS toolkit - Customize your experiments!
You want to have a maximum of activation or repression of your genes by a minimal effort? Then you
have to use the uniCAS toolkit provided by the iGEM team Freiburg 2013. All you have to do is:
- Click yourself through the routine below
- Order the appropriate plasmids and oligos
- Conduct a minimal of cloning
- Start your personalized experiment
