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<b> 1. Preparation of starting plasmids </b> | <b> 1. Preparation of starting plasmids </b> | ||
<br> | <br> | ||
+ | <div style="text-align: justify; margin-left:20px"> | ||
+ | 1. Preparation restriction of components </div> | ||
− | <div style="text- | + | <div style="text-align: justify; margin-left:40px"> |
+ | 1. Restriction using biobrick assembly enzymes <br> | ||
− | + | 2. This preparation step is needed to create sticky ends on the cassettes <br> | |
− | + | 3. This step is only performed once <br> | |
− | + | 4. Restriction with EcoRI and PstI (see restriction protocol) for all components </div> | |
− | + | ||
− | + | ||
<br> | <br> | ||
− | <div style="text- | + | <div style="text-align: justify; margin-left:20px">2. Ligation of cassettes into plasmids </div> |
− | <div style="text- | + | <div style="text-align: justify; margin-left:40px"> |
+ | 1. Ligation using T4-ligase (see ligation protocol)</div> | ||
<br> | <br> | ||
<style type="text/css"> | <style type="text/css"> | ||
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− | <div style="text- | + | <div style="text-align: justify; margin-left:20px">3. Final preparation steps </div> |
− | <div style="text- | + | <div style="text-align: justify; margin-left:40px"> |
− | + | 1. Transformation of 9 different combinations into competent cells (see transformation protocol)<br> | |
+ | 2. Selection with corresponding antibiotics</div> | ||
<br> <br> | <br> <br> | ||
− | <div style="text- | + | <div style="text-align: justify; margin-left:20px">4. Next day</div> |
− | <div style="text- | + | <div style="text-align: justify; margin-left:40px"> |
+ | 1. Colony pcr and gel run to check for sizes of cassettes <br> | ||
− | + | 2 . Miniprep and check concentration via nanodrop <br> | |
− | + | 3. Many aliquots needed (small volume because thawing time) for future reactions!</div> | |
<br> | <br> | ||
<br> | <br> | ||
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<br> <br> | <br> <br> | ||
<div style="text-indent:20px;">1. Good to know </div> | <div style="text-indent:20px;">1. Good to know </div> | ||
− | <div style="text- | + | <div style="text-align: justify; margin-left:40px"> |
+ | 1. The 3-A-assembly will be used to add more and more cassettes in a row <br> | ||
− | + | 2. It is important to only combine cassettes with the same number</div> <div style="text-indent:60px;">(1, 2 and 3 have varying spacer length)<br> | |
− | + | 3. We will add cassettes and test frequently for viability to determine the maximum</div><div style="text-indent:60px;"> target-sequence length<br> | |
− | + | 4. We want to combine about five cassettes </div> | |
<br> | <br> | ||
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− | <div style="text- | + | <div style="text-align: justify; margin-left:40px"> |
+ | 1. In each assembly cycle, there will be two cassettes (same number/length) added to one linearized plasmid <br> | ||
− | + | 2. In the first step, we can either just use the prepared plasmid with cassettes already inserted or use an empty one, because the part in between will be cut out anyway <br> | |
− | + | 3. The be able to select for plasmid with higher cassette content the resistances will cycle<br> | |
− | + | 4. The resistance cycle (for plasmids) is K C A <br> | |
− | + | 5. For the inserts, the resistance signals from which plasmid they will be cut<br> | |
− | + | 6. The plasmids resistance determines the selection antibiotics for that step!</div> | |
<br> | <br> | ||
<div style="text-indent:20px;">3. Legend </div> | <div style="text-indent:20px;">3. Legend </div> | ||
− | <div style="text- | + | <div style="text-align: justify; margin-left:20px"> |
− | < | + | CX – Chloramphenicol (Insert/Plasmid) from step X |
− | + | <br> | |
− | < | + | AX – Ampicillin (Insert/Plasmid) from step X |
− | + | <br> | |
− | < | + | KX – Kanamycin (Insert/Plasmid) from step X |
− | + | <br> | |
− | < | + | E – EcoRI |
− | + | <br> | |
− | < | + | S – SpeI |
− | + | <br> | |
− | < | + | X – XbaI |
− | + | <br> | |
+ | P – PstI | ||
</div> <br> | </div> <br> | ||
<style type="text/css"> | <style type="text/css"> | ||
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<div style="text-indent:20px;">4. Starting a cycle</div> | <div style="text-indent:20px;">4. Starting a cycle</div> | ||
− | <div style="text- | + | <div style="text-align: justify; margin-left:20px"> |
+ | 1. Insert 1 is cut out of plasmid C1/Insert 2 is cut out of plasmid A1 and plamsid K1 is linearized <br> | ||
− | + | 2. Insert 1 and 2 are ligated into plasmid K1 <br> | |
+ | 3. Insert 1 is cut out of plasmid A2/Insert 2 (here the fusion from Insert 1+2 from the first cycle) is cut out of K1 and plasmid C2 is linearized <br> | ||
+ | 4. Insert 1 and 2 are ligated into plasmid C2<br> | ||
− | + | 5. With insert 1 coming from K3 and insert 2 from C2 (fusion) the steps will repeated until maximum number of inserts is reached <br> | |
− | |||
− | + | 6. The assembly needs to be done for all 3 cassettes simultaneously after each step | |
− | + | <br> | |
− | + | 7. Transformation of ligation into competent cells (see transformation protocol) | |
− | + | <br> | |
− | < | + | 8. Selection with corresponding antibiotics </div> |
− | + | ||
− | < | + | |
− | + | ||
<br> | <br> | ||
<div style="text-indent:20px;">5. Next day</div> | <div style="text-indent:20px;">5. Next day</div> | ||
− | <div style="text- | + | <div style="text-align: justify; margin-left:20px"> |
− | < | + | 1. Colony pcr and gel run to check for sizes of cassettes |
− | + | <br> | |
+ | 2. Miniprep and check concentration via nanodrop</div> | ||
<br> <br> | <br> <br> | ||
− | + | ||
</div></div></div> | </div></div></div> | ||
Revision as of 09:46, 30 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols
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