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Nanodropinstrument

Manufactured by GE Healthcare
Sourced in United States

The NanoDropinstrument is a spectrophotometer that measures the concentration and purity of DNA, RNA, and protein samples in small volumes. It can analyze samples as small as 0.5 microliters without the need for cuvettes or other sample containment devices.

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2 protocols using nanodropinstrument

1

Plasmid Isolation and Sequence Verification

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Plasmids (Table S2, Supporting Information) were isolated using the
QIAprep Spin Miniprep Kit (Qiagen). Custom primers were obtained from
Integrated DNA Technologies (Table S3, Supporting
Information
). PCR reagents were purchased from Takara, New
England Biolabs, or Stratagene, and PCR products were isolated using
a QIAquick PCR Purification Kit (Qiagen). DNA fragments were isolated
from 0.7% agarose gels using the QIAquick Gel Extraction Kit (Qiagen).
All other DNA-modifying enzymes were purchased from New England Biolabs
and used according to the manufacturer’s protocols. When necessary,
purity and yield of extracted DNA were monitored using a NanoDrop
instrument (GE Healthcare). To create pQlink plasmids used for the
coexpression of multiple genes, reagents purchased from Invitrogen
were used for ligation-independent cloning (LIC) and as previously
described.41 (link) All plasmids constructed in
this study were transformed into a chemically competent E.
coli
XL-1 Blue storage strain before transformation into
the strain used in experiments (Table S2, Supporting
Information
). Plasmid promoter and gene-insert sequences were
verified by DNA sequencing at the ICMB Core Facility at the University
of Texas at Austin.
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2

Overexpression and Purification of DHFR Mutants

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WT DHFR and all mutants used in this study were cloned into a pET24 expression vector and overexpressed in the BL21(DE3) pLys E. coli strain.
A single colony of the transformed E. coli carrying the wild type or mutation dhfr was cultured in Luria-Bertani liquid medium containing 50 μg/mL kanamycin (LB-kana) at 30°C overnight, and then inoculated to fresh LB-kana (1:100 dilution) and incubated again at 30°C. When the OD600 of the culture reached 0.6, isopropyl β-D-1-thiogalactopyranoside (final concentration, 0.4 mM) was added. Cultures were incubated for an additional 12–16 h at 25°C. The cells were then collected by centrifugation and disrupted by sonication. The recombinant proteins were purified with Ni-NTA Superflow (QIAGEN, U.S.) according to the manufacturer’s instructions. Then, the collected protein sample was run with Superdex 75pg Column and was desalted with the desalting Column in ÄKTA protein purification system (GE Healthcare, U.S.). The final concentration of the purified protein was determined using the BCA protein assay kit (PIERCE CHEMICAL, USA) or the NanoDrop instrument (GE Healthcare, U.S.).
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