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<html> | <html> | ||
| - | <b>Action points</b> | + | <b>Action points</b><br> |
| - | Development of wiki | + | Development of wiki<br> |
| - | Brainstorming | + | Brainstorming<br> |
| - | DNA assembly talk | + | DNA assembly talk<br> |
| - | Brainstorming | + | Brainstorming<br> |
| - | Presentation of the ideas | + | Presentation of the ideas<br> |
| - | <b>Day</b> | + | <b>Day</b><br> |
With the sad news that James is still in hospital we started to work again. After meeting with advisors on friday, on monday we were ready to brainstorm again about many more ideas. In the morning we went to queens lawn to enjoy a bit of sunshine to help us thinking. After a while some of the ideas were formed. At noon we had a talk about DNA assembly and a after a quick lunch we split into groups and continue developing our ideas (you can find them on the brainstorming page). Before finishing for the day, we all presented some of the points and ideas, discarded some and kept some for further investigation. Tomorrow we hope to see James among us and brainstorm some more. | With the sad news that James is still in hospital we started to work again. After meeting with advisors on friday, on monday we were ready to brainstorm again about many more ideas. In the morning we went to queens lawn to enjoy a bit of sunshine to help us thinking. After a while some of the ideas were formed. At noon we had a talk about DNA assembly and a after a quick lunch we split into groups and continue developing our ideas (you can find them on the brainstorming page). Before finishing for the day, we all presented some of the points and ideas, discarded some and kept some for further investigation. Tomorrow we hope to see James among us and brainstorm some more. | ||
| + | </html> | ||
| + | <html> | ||
| + | <br><br><b>DNA assembly presentation:</b><br><br> | ||
| + | DNA assembly can kill rpojects if done incorrectly. It takes around 3 days for 1 assembly: <br> | ||
| + | Day 1: Start cultures<br> | ||
| + | Day 2: Assemble<br> | ||
| + | Day 3: Screen (might take more than one day). <br><br> | ||
| + | Assembly day: <br> | ||
| + | Miniprep ~30 mins.<br> | ||
| + | Extract part ~75-135 mins.<br> | ||
| + | Purify part ~90 mins. <br> | ||
| + | Assemble (make flanking sequences single stranded and mix and anneal) ~75-135 mins. <br> | ||
| + | Transform ~60 mins. <br> | ||
| + | <b>~6.5 hours! That is being optimistic!</b><br><br> | ||
| + | Two techniques: <br> | ||
| + | Short Overlap (biobricks)- Maximum two parts at the same time. <br> | ||
| + | Long overlap (Gibson/User) - Safely assemble 5 parts, theoretically 10. <br><br> | ||
| + | Short: <br> | ||
| + | Miniprep --> Restriction digest --> Gel extract --> Ligate--> Transform<br> | ||
| + | All parts submitted must be in this format. Can use the parts from miniprep a maximum of around 4 times. <br> | ||
| + | ~6 bp overhang. Low Tm (~10 degrees celsius). Ligase works best at 37 degrees celsius though. Need to compromise between both these paramters. <br><br> | ||
| + | Long: <br> | ||
| + | Miniprep --> PCR --> DpnI digest + purify or gel extract --> Assembly reaction--> Transform<br> | ||
| + | PCR primer could work first time or take weeks to get to work due to possibilities of non-specific binding and other errors that can occur with an inadequate primer. <br> | ||
| + | Gibson ~40bp overhang while User ~20bp overhang. User require a uracil for every 7th base. Uracils are expensive bases to buy. <br> | ||
| + | Gibson still work for DNA fragments down to ~91bp. Need to be careful that they're not too much shorter. <br><br> | ||
| + | Part insulation: <br> | ||
| + | If 2 ORF's are next to each other one might transcribe into another. Solve through three methods: <br> | ||
| + | 1: Place a terminator in between. Problem is that if there are too many terminators with the same sequence, deletions might occur. Check Biofab IS website for different terminators.<br> | ||
| + | 2: Face parts away from each other. <br> | ||
| + | 3: Spacing them away from each other. However, read-through is very long. <br><br> | ||
| + | Reusable parts: <br> | ||
| + | Biobricks allows us to reuse parts easily. Can this be done with the Long overhang methods? <br> | ||
| + | Prefix and suffix attached to GOI. Use linkers. Depending on linker we attach one GOI to another in whatever orientation and order we want. <br><br> | ||
| + | Libraries: <br> | ||
| + | Three types: Mutants, Mutagenomics and Rational design. <br> | ||
| + | Sources: Other labs/registries, epPCR, degenerate synthesis. | ||
</html> | </html> | ||
Latest revision as of 11:58, 12 July 2011
Action points
Development of wiki
Brainstorming
DNA assembly talk
Brainstorming
Presentation of the ideas
Day
With the sad news that James is still in hospital we started to work again. After meeting with advisors on friday, on monday we were ready to brainstorm again about many more ideas. In the morning we went to queens lawn to enjoy a bit of sunshine to help us thinking. After a while some of the ideas were formed. At noon we had a talk about DNA assembly and a after a quick lunch we split into groups and continue developing our ideas (you can find them on the brainstorming page). Before finishing for the day, we all presented some of the points and ideas, discarded some and kept some for further investigation. Tomorrow we hope to see James among us and brainstorm some more.
DNA assembly presentation:
DNA assembly can kill rpojects if done incorrectly. It takes around 3 days for 1 assembly:
Day 1: Start cultures
Day 2: Assemble
Day 3: Screen (might take more than one day).
Assembly day:
Miniprep ~30 mins.
Extract part ~75-135 mins.
Purify part ~90 mins.
Assemble (make flanking sequences single stranded and mix and anneal) ~75-135 mins.
Transform ~60 mins.
~6.5 hours! That is being optimistic!
Two techniques:
Short Overlap (biobricks)- Maximum two parts at the same time.
Long overlap (Gibson/User) - Safely assemble 5 parts, theoretically 10.
Short:
Miniprep --> Restriction digest --> Gel extract --> Ligate--> Transform
All parts submitted must be in this format. Can use the parts from miniprep a maximum of around 4 times.
~6 bp overhang. Low Tm (~10 degrees celsius). Ligase works best at 37 degrees celsius though. Need to compromise between both these paramters.
Long:
Miniprep --> PCR --> DpnI digest + purify or gel extract --> Assembly reaction--> Transform
PCR primer could work first time or take weeks to get to work due to possibilities of non-specific binding and other errors that can occur with an inadequate primer.
Gibson ~40bp overhang while User ~20bp overhang. User require a uracil for every 7th base. Uracils are expensive bases to buy.
Gibson still work for DNA fragments down to ~91bp. Need to be careful that they're not too much shorter.
Part insulation:
If 2 ORF's are next to each other one might transcribe into another. Solve through three methods:
1: Place a terminator in between. Problem is that if there are too many terminators with the same sequence, deletions might occur. Check Biofab IS website for different terminators.
2: Face parts away from each other.
3: Spacing them away from each other. However, read-through is very long.
Reusable parts:
Biobricks allows us to reuse parts easily. Can this be done with the Long overhang methods?
Prefix and suffix attached to GOI. Use linkers. Depending on linker we attach one GOI to another in whatever orientation and order we want.
Libraries:
Three types: Mutants, Mutagenomics and Rational design.
Sources: Other labs/registries, epPCR, degenerate synthesis.
