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• 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.