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Team:Cambridge-JIC/Dry Work
From 2014.igem.org
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| - | {{:Team:Cambridge-JIC/Templates/ | + | {{:Team:Cambridge-JIC/Templates/header_prototype2}} |
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| + | <div align="center"><a href="https://2014.igem.org/wiki/index.php?title=Team:Cambridge-JIC/Dry_Work&action=edit">Edit this page</a></div> | ||
<h3>Constructs</h3> | <h3>Constructs</h3> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Delete 5'-UTRs (if in doubt). 5'-UTR's affect expression rates, so if we're comparing promoters/inputs, UTR's will make a big difference. So, for the sake of normalisation, get rid of the 5'-UTRs.</li> |
| - | <li> | + | <li>If it works, don't change it. Don't go altering spacing in between parts when making constructs!</li> |
</ul> | </ul> | ||
| - | <h3>Primers</h3> | + | <h3>Primers, Gibson flaps and splitting up constructs</h3> |
<ul> | <ul> | ||
| - | <li> | + | <li>With our hi-tec polymerase, each fragment you're PCR'ing up can be up to 5.5kb (reliably). Any more, see if you can re-jig primers to get smaller fragments. i.e. 6kb - 2kb-2kb is not ideal, but 3kb - 4kb - 3kb is fine (or even 5kb-5kb) </li> |
| - | <li> | + | |
| + | <li>If you're splitting up your constructs, you don't need to add flaps - just choose primers appropriately!</li> | ||
| + | |||
| + | <li>Make the primers. Don't really worry about secondary structures unless it's obvious. Use a melting temperature of >58 degrees.</li> | ||
| + | |||
| + | <li>Check that they're correct by searching in the plasmid you're PCR'ing off and the destination construct. Make sure the primer can only prime to 1 place! (e.g. be careful using a nosT/35s region as these are very common) </li> | ||
| + | |||
| + | <li>Primers are '''cheap'''. If it's too much effort to reuse primers with your new construct, just order new ones. Spend some, but not a lot, of time if you're thinking of reusing primers to try and save a bit of dollar. </li> | ||
| + | |||
| + | <li>Try and make your primers reusable, if they're for a reusable part. Don't just split up your construct in any-which-way for Gibson. </li> | ||
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</ul> | </ul> | ||
</html> | </html> | ||
Latest revision as of 16:28, 29 July 2014
Constructs
- Delete 5'-UTRs (if in doubt). 5'-UTR's affect expression rates, so if we're comparing promoters/inputs, UTR's will make a big difference. So, for the sake of normalisation, get rid of the 5'-UTRs.
- If it works, don't change it. Don't go altering spacing in between parts when making constructs!
Primers, Gibson flaps and splitting up constructs
- With our hi-tec polymerase, each fragment you're PCR'ing up can be up to 5.5kb (reliably). Any more, see if you can re-jig primers to get smaller fragments. i.e. 6kb - 2kb-2kb is not ideal, but 3kb - 4kb - 3kb is fine (or even 5kb-5kb)
- If you're splitting up your constructs, you don't need to add flaps - just choose primers appropriately!
- Make the primers. Don't really worry about secondary structures unless it's obvious. Use a melting temperature of >58 degrees.
- Check that they're correct by searching in the plasmid you're PCR'ing off and the destination construct. Make sure the primer can only prime to 1 place! (e.g. be careful using a nosT/35s region as these are very common)
- Primers are '''cheap'''. If it's too much effort to reuse primers with your new construct, just order new ones. Spend some, but not a lot, of time if you're thinking of reusing primers to try and save a bit of dollar.
- Try and make your primers reusable, if they're for a reusable part. Don't just split up your construct in any-which-way for Gibson.
