Gold chloride

Figure 1 illustrates the highly simplified procedure of our FastCloning method. Briefly, after gel confirmation of PCR products, the remaining unpurified PCR reactions, containing amplified vector and insert, are mixed and digested with DpnI for 1 hour at 37°C. The digested mixture is then directly transformed into chemically competent Stratagene XL-10 Gold or NEB 10-beta E. coli cells.
The primers were designed with Oligo Analyzer 1.5 (http://www.genelink.com) to have an annealing temperature around 60°C (Nearest Neighbor method). The forward primer for vector amplification is in the 3' side of the polylinker region. The reverse primer for vector amplification is in the 5' side of the polylinker, and its reverse and complementary sequence was generated by Oligo Explorer 1.5. The primers for insert amplification have insert-specific sequences and additional 15-17 bases (depending on the GC content) overlapping with the vector ends. The specific sequences of primers used in this study are listed in Table 1. All the primers used in this study were synthesized by Invitrogen Corporation (Carlsbad, CA).
The PCR reaction components were: 50 μl total volume, 0.5 μl Phusion DNA polymerase (New England Biolabs, Ipswich, MA), or 0.8 μl Pfu Turbo, or PfuUltra DNA polymerase (Agilent Technologies, Inc, Santa Clara, CA), 5 μl 10× buffer; 5 μl of 2.5 mM dNTPs; 10 ng of plasmid DNA template; 5 pmol of each primer. The PCR cycling parameters were 98°C 3 min, (98°C 10 sec, 55°C 30 sec, 72°C 20 sec/kb) × 18 cycles, 72°C 5 min, and 4°C infinite for Phusion DNA polymerase, and 95°C 3 min, (95°C 15 sec, 55°C 1 min, 72°C 1 min/kb) × 18 cycles, 72°C 5 min, and 4°C infinite for Pfu Turbo or PfuUltra DNA Polymerase. The PCR products (5 μl for each product) were examined with 1% agarose gel electrophoresis with ethidium bromide staining using VWR Mini Gel electrophoresis setup (VWR International, Marietta, GA, USA) running at 100 V for 30 min. The PCR products were then visualized under a UV transilluminator, and gel pictures were taken using an AGFA scanner.
After confirmation of PCR products, 1 μl of DpnI enzyme (New England Biolabs) was added into the remaining unpurified PCR reactions (45 μl for each product) for vector or insert separately. The vector and insert were then mixed with 1:1 ratio (1:1, 1:2, and 1:4 for α9 nAChR subunit), and digested at 37°C for 1 hour. Two micro-liters (2, 4, and 8 μl for α9 nAChR) of the digested vector-insert mixture were then added to 40 μl of chemically competent XL-10 Gold E. coli cells (prepared with rubidium chloride method) unless indicated otherwise. The mixture was then incubated for 30 min on ice. After heat shock at 42°C for 45 sec, 350 μl of SOC medium was added to the mixture. After 60 min shaking at 37°C and 350 rpm with an Eppendorf Thermomixer, the entire content was plated onto the LB agar plate containing 100 μg/ml ampicillin. The plates were then incubated at 37°C overnight. Next day, colonies from each constructs were picked for PCR confirmation of each construct using GoTaq DNA polymerase (Promega, Madison, WI, USA) and vector specific primers, and also for inoculation in the LB medium (with ampicillin) for overnight culture of each clone for mini-prep. The DNA mini-prep was performed using QIAprep Spin Miniprep Kit (QIAGEN, Valencia, CA, USA). All the cloned sequences were finally confirmed by automated DNA sequencing at the DNA lab of the Arizona State University using primers in the vectors.
Figure 2 illustrates the application of this method to construct cDNAs encoding chimeric or fusion proteins. In this case, the PCR amplification of the vector also includes part of the cDNA in both ends. To insert a cDNA encoding a full-length protein, such as green fluorescence protein, the insert amplification should cover the entire coding region of the cDNA. For homologous domain swap chimera, insert amplification only needs to cover the corresponding region of a cDNA encoding a homologous fragment of protein. The detailed experimental procedures for chimera construction are essentially the same as cloning.
For insertion (or substitution) of a short DNA fragment (< 120 bp), the insert can be directly included in the primer sequences for vector amplification (Figure 3). This is a very convenient way to insert a short tag (such as myc tag or FLAG-tag) or to replace a short DNA fragment for chimera construction without limitation of the availability of specific DNA template for the insert. Introducing multiple mutations in a short (up to 120 bp) stretch of a cDNA is equivalent to replacing a short DNA fragment for chimera construction.

For transmission electron microscopy, freshly isolated exosome suspensions were fixed in 4% paraformaldehyde for 1 hour. Exosome suspensions from different samples (approximately 5 μl) were applied to copper mesh Formvar coated carbon stabilized grids, were allowed to adsorb to the grid for 4–5 minutes and then were wicked off with filter paper. For negative staining of exosomes, 1% Aqueous Uranyl Acetate (5 μl) was applied to the grid for 30 seconds, then wicked off with Whatman filter paper. Grids were allowed to thoroughly dry before viewing.
As for immunoelectron labelling with anti-CD63 and anti-CD9, exosome samples were fixed overnight in 4% paraformaldehyde diluted in 0.1M cacodylate buffer (pH 7.4). Fixed exosome preparations (20 μl) were applied to a carbon-Formvar coated 200 mesh nickel grids, and samples were allowed to stand for 30 minutes before wiping off excess using Whatman filter paper. Grids were then floated (sample side down) onto a 20 μl drop of 1M Ammonium Chloride for 30 minutes to quench aldehyde groups from the fixation step, followed by floating on drops of blocking buffer (0.4% BSA in PBS) for 2 hours. Grids were rinsed 3 times (5 minutes each) using 1xPBS and then were allowed to incubate with either blocking buffer only (negative control) or primary antibody (CD63) diluted with blocking buffer (1:100) for 1 hour. Rinsing of the grids using deionized water (3 times for 5 minutes each) and 1xPBS followed the incubation step. Grids were then floated on drops of 1.4 nm anti-rabbit nanogold (Nanoprobes, Inc.) diluted 1:1000 in blocking buffer for 1 hour. Enhancing of grids using HQ Silver (gold enhancement reagent, Nanoprobes, Inc.) was then performed for 1 minute, followed by rinsing in deionized ice-cold water. As a final step, negative staining in 2% aqueous Uranyl Acetate was performed, and samples were wicked dry and then allowed to air dry. TEM examination was performed using JEM 1230 transmission electron microscope (JEOL USA Inc., Peabody, MA) at 110 kV and imaged with an UltraScan 4000 CCD camera & First Light Digital Camera Controller (Gatan Inc., Pleasanton, CA). TEM sample preparation and imaging was performed at the Electron Microscopy and Histology Core Laboratory at Augusta University (www.augusta.edu/mcg/cba/emhisto/).

The colloidal gold solution (in-house) was prepared by adding 90 mL of distilled water into a triangular flask and heating it to 100°C while stirring on a hot stirrer plate (DAIHAN Scientific, Wonju, Republic of Korea). Once the temperature reached 100°C, 1% (w/v) gold chloride trihydrate (10 mL) was added and dissolved. After the gold chloride trihydrate solution was completely dissolved, 1 mL of 1% sodium citrate tribasic dehydrate was added and dissolved for 10 min at room temperature until the color of the reaction solution changed from yellow to the final red color. The resulting colloidal gold solution was diluted with distilled water, and the absorbance was measured using a UV-1650PC spectrophotometer (Shimazu, Tokyo, Japan).

The cells-free supernatant of S. albogriseolus was used to synthesize GNPs. A stock solution of 1000 μg/mL gold (III) was prepared by dissolving precise amount of Gold (III) chloride trihydrate (HAuCl₄.3H₂O) in distilled water. The cells-free supernatant of S. albogriseolus was mixed with varying concentrations of HAuCl₄ solution at a 1:2 volume-to-volume ratio, and the mixture was then incubated at 37 °C in the dark for 24–96 h. After the incubation period, the mixture's color changed to red or dark purple as a result of the reduction of gold chloride and GNPs formation. As a control, the cells-free supernatant was used without the addition of HAuCl₄ solution.