Equipment and materials
1. GDS-80 gene gun with 4.5 mm barrel, pressure regulator and hose (WGB09O001) (Wealtec)
2. Aseptic table
3. 3 cm target spacer (Wealtec)
4. Purity 99.999% helium
5. Sample: Zea mays L. embryo.
Experimental procedure
1. Adjust the GDS-80 gene gun output pressure setting and adjust the settings as indicated in the instructions before the experiment to ensure uniform bombardment. Note that after adjusting the uniform spray conditions, the sample cannula and barrel are cleaned with 70% ethanol.
2. The aseptic table should be cleaned and disinfected before operation. (At least UV exposure for half an hour)
3. Sterilize the barrel, sample cannula, target spacer, and forceps before placing them in the aseptic table.
4. Spray 70% ethanol on the surface of the GDS-80 gene gun body and hose assembly before placing them on the aseptic table.
5. Assemble the tube and sample loading tube so that the GDS-80 gene gun body is placed inside the aseptic table and connected to the entire GDS-80 system.
6. Set the pressure regulator to 50 psi and ensure that the gas flow rate is approximately 10-15 L/min.
7. Prepare the DNA plasmid/gold powder solution prior to bombardment. (0.5 ug DNA / 0.148 mg gold powder)
8. Bombard the sample with the aid of a 3 or 6 cm target spacer and transfer the sample to the MS medium after bombardment.
9. Incubate the sample for at least two days and then stain with a GUS staining solution for one day.
10. Observe the results under a microscope.
result
(a)
(b)
Figure 1. Introduction of the GUS gene into corn germ at 50 psi with the aid of a 3 cm target spacer.
(a) in normal conditions without pollution (b) in contaminated media
discuss
When operating in an aseptic workstation, there are many aspects that need to be taken care of at all times, otherwise it may cause serious contamination and affect the observation of the experiment (Figure 1). If the inside of the dish is contaminated, even if the DNA sample is not contaminated, the GUS protein content will be too small to be detected.
Pollution is usually caused by the following conditions, and proper attention can be avoided:
a. Pollution caused by bombardment equipment.
When operating the GDS-80 gene gun on an aseptic table, it is easy for the user to forget to disassemble the barrel and the sample loading sleeve for disinfection, and only spray 70% of the alcohol on the outer surface of the sample loading sleeve. Such disinfection is not complete, because even if the sample of the bombarded raw DNA plasmid is prepared in 100% pure alcohol, it is still difficult to remove the microorganisms attached to the barrel and the sample loading sleeve, thus making the bacteria in the transfer process. Will be pushed together and spread to the sample. The GDS-80 gene gun can be used in plant-specific experiments to assist in better experimentation, for example, using target spacers to aid tissue experiments in culture dishes, or using UTS-10 for callus cell experiments. All the parts used in the experiment, even the parts that are used as supporting samples, are easily accessible to the sample, so these parts should be adequately sterilized to prevent contamination. All parts of the laboratory equipment can be sterilized at 121 ° C for 30 minutes using an autoclave to ensure thorough disinfection. Another method of disinfection is to place the barrel and sample loading sleeve in a 70% ethanol solution for more than 20 minutes, dry it and place it in an aseptic table.
b. Contamination from the sample itself.
The plant samples taken from the field are in contact with many microorganisms. If the sample is not disinfected before the experiment, it will be easily contaminated in the medium, so the sample must be disinfected before the experiment. Due to the different characteristics of the samples, the method of disinfection is different. The most common method is to soak the sample in the prepared solution and add the appropriate proportion of bleach and Tween-20, soak it together for more than 20 minutes, then wash it with sterile distilled water and disperse the sample in the clean before the experiment. Dry in a petri dish.
c. Unskilled operation can result in contamination in the aseptic workstation.
When conducting experiments inside the aseptic console, most people will ignore some important links. Before performing the experiment in the aseptic workstation, be sure to wash your hands thoroughly and disinfect with 70% ethanol. Once you have entered the aseptic table for both hands, do not remove your hand from the console before ending the experiment. If there is a need to move the object in or out of the aseptic table, be sure to disinfect the hands with 70% ethanol before returning to the inside of the aseptic table. When operating the culture dish, do not open the cover too much, and do not let the hand or any object pass over it when the cover is open. Make sure to sterilize the pipette tip before sending it to the aseptic table. Replace the pipette tip before each sample is taken. You can only use the tweezers to process the target sample, and also ensure that the tweezers are sterilized with fire before each operation, and that the tweezers are not allowed to touch anything other than the sample. If the sample falls on the table, do not pick it up and put it back in the medium. Make sure that all equipment and materials have been disinfected using the correct method before placing them on the aseptic workstation.
d. The aseptic table is not well maintained.
Depending on the design of the aseptic table, the filter in the station should be replaced after a period of use, or when the effect of the filtrate on the microorganisms is reduced. Please periodically replace the filters in the aseptic table to maintain an optimal sterile environment. Even if the aseptic table is well maintained, the operator still needs to spray a 70% ethanol solution to clean the operating area inside the machine before use.
All laboratory equipment must be sterilized strictly prior to overall system operation. No contamination is allowed during operation. Although the standard operating procedures (SOPs) of each laboratory's sterile laboratory are different, the main objectives are the same, all based on pollution prevention. The operator should follow the SOP carefully and pay attention to several important aspects of how to avoid microbial contamination.
Forklift Caster
Forklift Caster
Ningbo Mywin Caster Co., Ltd. , https://www.mywin-caster.com