Team:UCLA/Notebook/Protein Cages/29 June 2015

iGEM UCLA




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.

Resuspending Newly-Synthesized DNA

Source: 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