Team:Cambridge-JIC/Dry Work

From 2014.igem.org

(Difference between revisions)

Latest revision as of 16:28, 29 July 2014

Edit this page

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.

@@ Line 1: / Line 1: @@
-{{:Team:Cambridge-JIC/Templates/header}}
+{{:Team:Cambridge-JIC/Templates/header_prototype2}}
 <html>
+<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>
 <ul>
-<li>Tip 1</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>
-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>If it works, don't change it. Don't go altering spacing in between parts when making constructs!</li>
-<li>Tip 2</li>
-If it works, don't change it. Don't go altering spacing in between parts when making constructs!
 </ul>
-<h3>Primers</h3>
+<h3>Primers, Gibson flaps and splitting up constructs</h3>
 <ul>
-<li>Tip 1</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>
-Make the primers. Don't really worry about secondary structures unless it's obvious. Use a melting temperature of >58 degrees.
-<li>Tip 2</li>
+<li>If you're splitting up your constructs, you don't need to add flaps - just choose primers appropriately!</li>
-Check that they are correct by searching in the plasmid you're PCR'ing off and the destination construct.
+<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>
 </ul>
 </html>