Difference between revisions of "Team:UCLA/Notebook/Protein Cages/29 June 2015"
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Intro: Today I delved into the specifics of resuspending newly synthesized DNA once we receive it from IDT. Phil and I discussed primer design in more detail. We will order the cage gene flanked by the iGEM prefix and suffix, then use one round of PCR with primers overlapping the cage gene itself and including the NdeI and XhoI sites. Additionally, I designed amplification primers for the cage gene itself. Phil and I researched which sequencing primers to use for the pET22b vector. | Intro: Today I delved into the specifics of resuspending newly synthesized DNA once we receive it from IDT. Phil and I discussed primer design in more detail. We will order the cage gene flanked by the iGEM prefix and suffix, then use one round of PCR with primers overlapping the cage gene itself and including the NdeI and XhoI sites. Additionally, I designed amplification primers for the cage gene itself. Phil and I researched which sequencing primers to use for the pET22b vector. | ||
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https://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2011/03/16/dna-oligonucleotide-resuspension-and-storage | https://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2011/03/16/dna-oligonucleotide-resuspension-and-storage | ||
Revision as of 22:38, 29 June 2015
Intro: Today I delved into the specifics of resuspending newly synthesized DNA once we receive it from IDT. Phil and I discussed primer design in more detail. We will order the cage gene flanked by the iGEM prefix and suffix, then use one round of PCR with primers overlapping the cage gene itself and including the NdeI and XhoI sites. Additionally, I designed amplification primers for the cage gene itself. Phil and I researched which sequencing primers to use for the pET22b vector.
https://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2011/03/16/dna-oligonucleotide-resuspension-and-storage lyophilized (freeze-dried) spin down prior to opening use TE buffer Tris-EDTA pH 8.0 (nuclease free) store at -20C conc notes: 5-10 mM usually highest possible without precipitation 100 uM stock is what IDT does, then dilutes a portion to work with Q5 PCR protocol calls for 1 ng-1ug of genomic DNA and 1pg-1ng of plasmid/viral DNA (NEB) https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491