Difference between revisions of "Team:UCLA/Notebook/Protein Cages/29 June 2015"
| Line 8: | Line 8: | ||
lyophilized (freeze-dried) | lyophilized (freeze-dried) | ||
| + | |||
spin down prior to opening | spin down prior to opening | ||
| + | |||
use TE buffer | use TE buffer | ||
| + | |||
Tris-EDTA pH 8.0 (nuclease free) | Tris-EDTA pH 8.0 (nuclease free) | ||
| + | |||
store at -20C | store at -20C | ||
| + | |||
conc notes: | conc notes: | ||
| + | |||
5-10 mM usually highest possible without precipitation | 5-10 mM usually highest possible without precipitation | ||
| + | |||
100 uM stock is what IDT does, then dilutes a portion to work with | 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 | + | |
| + | 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 | ||
Revision as of 22:40, 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.
Resuspending Newly-Synthesized DNA
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