Particle bombardment was conducted as described previously (11 (link)) using Biolistic Particle Delivery System (Bio-Rad) PDS-1000. The upper epidermal layer of the onion was peeled and placed on a plate containing half-strength MS medium (0.25% sucrose). A total of 3.5 μg of each plasmid DNA was used, and the gold particles size was 1.0 μm (Inbio Gold). There were a total of 0.3–0.4-mg particles per shot, and the particles were accelerated with a pressure of 1,100 psi. The distance from the projectile source to the sample was 6 cm. After bombardment, the onion epidermal layers were incubated at room temperature in the dark for 16 h before observation.
Biolistic particle delivery system
Manufactured by Bio-Rad
Sourced in United States
The Biolistic Particle Delivery System is a laboratory equipment designed for the transfection of cells, tissues, or organisms with genetic material. The system utilizes a pressurized gas to accelerate DNA-coated microparticles into the target sample, allowing for the introduction of genetic material. The core function of the Biolistic Particle Delivery System is to facilitate the delivery of genetic material into cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse te300, by Nikon (2 mentions)
Pds 1000, by Bio-Rad (1 mentions)
Pds 1000 he system, by Bio-Rad (1 mentions)
Pds 1000 he, by Bio-Rad (1 mentions)
Polyethylene glycol 4000, by Merck Group (1 mentions)
Las af, by Leica (1 mentions)
Tcs sp5 system, by Leica (1 mentions)
Dm6000b, by Leica (1 mentions)
7 protocols using biolistic particle delivery system
1
Onion Epidermal Transient Transformation
2
Transient Expression of MiMsp40 in Onion Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The MiMsp40 gene with and without signal peptide-encoding regions was amplified using the gene-specific primer pairs M40-F1 KpnI/M40- R1 XbaI and M40-F2 KpnI/M40- R1 XbaI, containing KpnI and XbaI restriction enzyme sites in the forward and reverse primers, respectively (Supplemental Table S2 ). The resulting amplified fragments were cloned into the respective sites in a modified p35SeGFP vector between the cauliflower mosaic virus (CaMV) 35S promoter and enhanced green fluorescent protein (eGFP) to express the eGFP fusion protein; the p35SeGFP vector without MiMsp40 was used as a control. Both constructs were confirmed by DNA sequencing. The resulting constructs were introduced into onion (Allium cepa) epidermal cells by biolistic bombardment with a PDS1000/He system (Biolistic Particle Delivery System, Bio-Rad, CA, USA). Onion pieces were incubated for 24 h at 24 °C in the dark; epidermal peels were then observed through a fluorescence microscope (Nikon Eclipse TE300, Tokyo, Japan).
3
Particle Bombardment of Onion Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmids for particle bombardment (pUGW2/XXX, ptd/MEB2, and pBI221/SP-GFP-HDEL) were bombarded into onion epidermis cells using the Biolistic Particle Delivery System (Bio-Rad Laboratories) according to the manufacturer’s instructions.
4
Visualizing Annexin Localization in Onion Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
Ha-annexin ORF sequences (without a stop codon) were amplified using primers ann-Xba I-S and ann-Xho I-AS (S1 Table ) for cloning into the pUC35SGFP vector to generate the 35S:ANNEXIN:GFP construct by the method of digestion and connection. Each construct plasmid (4–5 μg/shot) was delivered into onion epidermal cells through biolistic bombardment by vacuumizing to 27–28 in. Hg using a PDS1000/He system (Biolistic Particle Delivery System, Bio-Rad, USA). After bombardment, epidermal peels were incubated at 25°C for 24 h in the dark. The subcellular localization of the fused proteins was visualized using laser confocal fluorescence microscopy (Nikon Eclipse TE300, Nikon, Japan) at an excitation wavelength of 488 nm.
+ Expand
Ha-annexin ORF sequences (without a stop codon) were amplified using primers ann-Xba I-S and ann-Xho I-AS (
5
Dual-Luciferase Reporter Assay in Arabidopsis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gold particles (3 μg) were prepared for each bombardment. Gold particles were washed in 70% ethanol and sterile water and resuspended in 50% sterile glycerol. Five microgram of each Prom::GreenLUC-pGREEN and 35Sprom::RedLUC-pJAN DNA were mixed with 50 μl gold beads, 50 μl 2.5 M CaCl2 and 20 μl 0.1 M spermidine. After two washes with 100% ethanol, the DNA–gold mix was resuspended in 50 μl 100% ethanol and used for two parallel bombardments of 5–10 mm Arabidopsis leaves using the Biolistic Particle Delivery System (Bio-Rad, PDS-1000/HE). After 12–24 h incubation in LD, luciferin (1 mM) was sprayed on the leaves and the emitted light was immediately measured with a charge-coupled device-camera adapted with optical filters to detect RedLUC or GreenLUC signals independently. The ratio of GreenLUC/RedLUC signals was calculated with the excel macro Chroma-LUC Calculator version 1.0 (Promega).
6
Subcellular Localization of Arabidopsis ALDH10s
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
N- and C- terminal GFP-fusion plasmids containing one of the Arabidopsis ALDH10s co-transformed with one of mCherry peroxisomal (pRTL2/Cherry-PTS1)37 (link), RFP cytosolic (pRTL2-MCS-RFP-stop)31 or RFP plastidial38 (link) markers. A ratio of 8:2 (μg GFP:mCherry or RFP) was used for the two plasmid constructs. Protoplasts were prepared from Arabidopsis cell suspension culture ecotype Col-0 as described previously39 . Polyethylene glycol 4000 (Sigma, 81240) was utilized for protoplast transformation as described previously40 41 42 with some minor modifications. Also, onion epidermal segments were peeled and placed on an MS plate (inner side up) for transient co-transformation using a biolistic particle delivery system (BioRad Laboratories). Onion cells or Arabidopsis protoplasts were incubated for 16 h in the dark at 24 °C prior to microscopy and then examined using an upright Leica DM 6000B confocal laser scanning microscope connected to a Leica TCS SP5 system. Argon and HeNe 542 lasers were activated for visualization of GFP and RFP or mCherry fluorescent proteins. The excitation/emission scan settings were 488 nm/505 nm for the mGFP channel and 543 nm/610 nm for the RFP/mcherry channel. Modulation of laser light intensity and time-lapse scanning were performed using the Leica software LAS AF.
7
Sugarcane Callus Transformation via Biolistic
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The osmoticum medium was prepared using CIM3 supplemented with sorbitol and Mannitol (0.2 M for both). The calli were transferred to the osmoticum medium 3 hours before bombardment. The induced calli of sugarcane were bombarded with gold particles coated with the desired construct (pGreenII0129), at concentrations of 2.8, 5.6 and 8.5µg/µl, 1100 psi and vacuum pressure 25 in Hg using biolistic particle delivery system (Bio-Rad). Calli were then incubated at 28 o C for 48 hours.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!