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<div style="text-align: justify; margin-left:40px"> | <div style="text-align: justify; margin-left:40px"> | ||
− | 1. Restriction using biobrick assembly enzymes <br> | + | 1. Restriction using biobrick assembly enzymes. <br> |
− | 2. This preparation step is needed to create sticky ends on the cassettes <br> | + | 2. This preparation step is needed to create sticky ends on the cassettes. <br> |
− | 3. This step is only performed once <br> | + | 3. This step is only performed once. <br> |
− | 4. Restriction with EcoRI and PstI (see restriction protocol) for all components </div> | + | 4. Restriction with EcoRI and PstI (see restriction protocol) for all components. </div> |
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<div style="text-align: justify; margin-left:20px">3. Final preparation steps </div> | <div style="text-align: justify; margin-left:20px">3. Final preparation steps </div> | ||
<div style="text-align: justify; margin-left:40px"> | <div style="text-align: justify; margin-left:40px"> | ||
− | 1. Transformation of 9 different combinations into competent cells (see transformation protocol)<br> | + | 1. Transformation of 9 different combinations into competent cells (see transformation protocol).<br> |
− | 2. Selection with corresponding antibiotics</div> | + | 2. Selection with corresponding antibiotics.</div> |
<br> | <br> | ||
<div style="text-align: justify; margin-left:20px">4. Next day</div> | <div style="text-align: justify; margin-left:20px">4. Next day</div> | ||
<div style="text-align: justify; margin-left:40px"> | <div style="text-align: justify; margin-left:40px"> | ||
− | 1. Colony pcr and gel run to check for sizes of cassettes <br> | + | 1. Colony pcr and gel run to check for sizes of cassettes. <br> |
− | 2. Miniprep and check concentration via nanodrop <br> | + | 2. Miniprep and check concentration via nanodrop. <br> |
3. Many aliquots needed (small volume because thawing time) for future reactions!</div> | 3. Many aliquots needed (small volume because thawing time) for future reactions!</div> | ||
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<div style="text-indent:20px;">1. Good to know </div> | <div style="text-indent:20px;">1. Good to know </div> | ||
<div style="text-align: justify; margin-left:40px"> | <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> | + | 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 (1, 2 and 3 have varying spacer length)<br> | + | 2. It is important to only combine cassettes with the same number (1, 2 and 3 have varying spacer length).<br> |
− | 3. We will add cassettes and test frequently for viability to determine the maximum target-sequence length<br> | + | 3. We will add cassettes and test frequently for viability to determine the maximum target-sequence length.<br> |
− | 4. We want to combine about five cassettes </div> | + | 4. We want to combine about five cassettes. </div> |
<br> | <br> | ||
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<div style="text-align: justify; margin-left:40px"> | <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> | + | 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> | + | 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> | + | 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> | + | 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> | + | 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> | 6. The plasmids resistance determines the selection antibiotics for that step!</div> | ||
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<div style="text-align: justify; margin-left:40px"> | <div style="text-align: justify; margin-left:40px"> | ||
− | 1. Insert 1 is cut out of plasmid C1/Insert 2 is cut out of plasmid A1 and plamsid K1 is linearized <br> | + | 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> | + | 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> | + | 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> | + | 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> | + | 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 | + | 6. The assembly needs to be done for all 3 cassettes simultaneously after each step. |
<br> | <br> | ||
− | 7. Transformation of ligation into competent cells (see transformation protocol) | + | 7. Transformation of ligation into competent cells (see transformation protocol). |
<br> | <br> | ||
− | 8. Selection with corresponding antibiotics </div> | + | 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-align: justify; margin-left:40px"> | <div style="text-align: justify; margin-left:40px"> | ||
− | 1. Colony pcr and gel run to check for sizes of cassettes | + | 1. Colony pcr and gel run to check for sizes of cassettes. |
<br> | <br> | ||
− | 2. Miniprep and check concentration via nanodrop</div> | + | 2. Miniprep and check concentration via nanodrop.</div> |
<br> <br> | <br> <br> | ||
<hr size="10" noshade></hr> | <hr size="10" noshade></hr> | ||
− | <p style="font-size:15pt;" | + | <p style="font-size:15pt;"><sup>[1]</sup> |
http://parts.igem.org/Help:Protocols/3A_Assembly</p> | http://parts.igem.org/Help:Protocols/3A_Assembly</p> | ||
</div></div></div> | </div></div></div> |
Revision as of 14:37, 31 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols
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