Paulinchen (Talk | contribs) (protocols) |
Paulinchen (Talk | contribs) (protocols) |
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<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="3 A Assembly" /> | <input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="3 A Assembly" /> | ||
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+ | |||
+ | 2) Preparation of starting plasmids | ||
+ | |||
+ | |||
+ | Preparation restriction of components | ||
+ | |||
+ | |||
+ | - restriction using biobrick assembly enzymes | ||
+ | |||
+ | - this preparation step is needed to create sticky ends on the cassettes | ||
+ | |||
+ | - this step is only performed once | ||
+ | |||
+ | - restriction with EcoRI and PstI (see restriction protocol) for all components | ||
+ | |||
+ | |||
+ | Ligation of cassettes into plasmids | ||
+ | |||
+ | - ligation using T4-ligase (see ligation protocol) | ||
+ | |||
+ | Plasmid | ||
+ | cassettes | ||
+ | psB1C3 | ||
+ | 1 | ||
+ | 2 | ||
+ | 3 | ||
+ | psB1A3 | ||
+ | 1 | ||
+ | 2 | ||
+ | 3 | ||
+ | psB1K3 | ||
+ | 1 | ||
+ | 2 | ||
+ | 3 | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | Final preparation steps | ||
+ | |||
+ | - transformation of 9 different combinations into competent cells (see transformation protocol) | ||
+ | |||
+ | - selection with corresponding antibiotics | ||
+ | |||
+ | Next day | ||
+ | |||
+ | - colony pcr and gel run to check for sizes of cassettes | ||
+ | |||
+ | - miniprep and check concentration via nanodrop | ||
+ | |||
+ | - many aliquots needed (small volume because thawing time) for future reactions! | ||
+ | |||
+ | |||
+ | 3) 3A-assembly | ||
+ | |||
+ | - the 3-A-assembly will be used to add more and more cassettes in a row (see 3-A-assembly protocol) | ||
+ | |||
+ | - it is important to only combine cassettes with the same number (1, 2 and 3 have varying spacer length) | ||
+ | |||
+ | - we will add cassettes and test frequently for viability to determine the maximum target-sequence length | ||
+ | |||
+ | - we want to combine about five cassettes | ||
+ | |||
+ | |||
+ | Assembly scheme | ||
+ | |||
+ | - in each assembly cycle, there will be two cassettes (same number/length) added to one linearized plasmid | ||
+ | |||
+ | - 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 | ||
+ | |||
+ | - the be able to select for plasmid with higher cassette content the resistances will cycle | ||
+ | |||
+ | - the resistance cycle (for plasmids) is K C A | ||
+ | |||
+ | - for the inserts, the resistance signals from which plasmid they will be cut | ||
+ | |||
+ | - the plasmids resistance determines the selection antibiotics for that step! | ||
+ | |||
+ | |||
+ | Legend | ||
+ | |||
+ | - CX – Chloramphenicol (Insert/Plasmid) from step X | ||
+ | - AX – Ampicillin (Insert/Plasmid) from step X | ||
+ | - KX – Kanamycin (Insert/Plasmid) from step X | ||
+ | - E – EcoRI | ||
+ | - S – SpeI | ||
+ | - X – XbaI | ||
+ | - P – PstI | ||
+ | |||
+ | </div></div> | ||
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<input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="DNA Purification" /> | <input type="button" style="height: 50px; width: 50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="DNA Purification" /> | ||
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+ | |||
+ | Depending on the PCR product | ||
+ | Binding of DNA | ||
+ | 1. Insert SV Minicolumn into Collection Tube. | ||
+ | 2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate | ||
+ | at room temperature for 1 minute. | ||
+ | 3. Centrifuge at 16,000 ×g for 1 minute. Discard (verwerfen) flowthrough and reinsert Minicolumn | ||
+ | into Collection Tube. | ||
+ | Washing | ||
+ | 4. Add 700 μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. | ||
+ | Discard flowthrough and reinsert Minicolumn into Collection Tube. | ||
+ | 5. Repeat Step | ||
+ | 4 with 500 μl | ||
+ | Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes. | ||
+ | 6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the | ||
+ | microcentrifuge lid | ||
+ | open | ||
+ | (or off) to allow evaporation of any residual ethanol. | ||
+ | Elution | ||
+ | 7. | ||
+ | Carefully | ||
+ | transfer Minicolumn to a clean 1.5 ml microcentrifuge tube. | ||
+ | 8. Add 50 μl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for | ||
+ | 1 minute. Centrifuge at 16,000 × g for 1 minute. | ||
+ | 9. Discard Minicolumn and store DNA at 4°C or –20°C | ||
+ | |||
+ | </div></div> | ||
Revision as of 20:14, 25 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols
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