Methodology:A. Preparation of Overnight Bacterial Culture1. A single colony is inoculated by using autoclaved tips from the agar plate containing single colonies of Bacillus amyloliquefaciens UMAS 1002.
2. The tip with Bacillus amyloliquefaciens UMAS 1002 is added into bijou bottle containing 5 ml of LB media. The culture is then incubated with shaking for overnight at 370C.
3. The bacterial cultures are kept at 40C for used in further DNA analysis and isolation.
B. Isolation and Preparation of Genomic DNA from Bacteria1. One and a half of an overnight culture is transferred into Eppendorf tube and centrifuged for 30 seconds.
2. After centrifugation, the supernatant is removed and the cell pellet is resuspended in 567µL TE buffer. The cell pellet is mixed well by continuous pippeting.
3. 30µL of 10% (w/v) SDS and 3µL 20mg/ml Proteinase K is added to give a final concentration of 100µg/ml proteinase K in 0.5% SDS solution. 100 µL of 5M NaCl solutions is also added and mixed well.
4. 80µL of CTAB/NaCl is added into the mixture. The culture is mixed well and incubated for 10 minutes in water bath at 65¬0C.
5. After 10 minutes, 780µl of Phenol / Chloroform / Isoamyl alcohol (25: 24: 1) that is added to the mixture. Then, the mixture is vortexed briefly and centrifuged for 5 minutes to separate the phases.
6. The viscous and clear supernatant is transferred carefully into new Eppendorf tube. The aqueous DNA layer once again is re-extract using Chloroform/Isoamyl alcohol (24: 1) and centrifuged for 5 minutes.
7. After centrifugation, the supernatant is transferred into a new Eppendorf tube. 420µl of isopropanol is added to precipitate out the nucleic acid. The tube is vortexed up and down slowly until the white DNA precipitate appears. The DNA precipitate can be pelleted by centrifugation for 30 seconds.
8. The supernatant is removed and the DNA pellet is washed with 200µL of 70% ethanol and centrifuged for another 30 seconds at room temperature. The supernatant is carefully removed and the pellet is air-dry.
9. The dried DNA pellet is dissolved in 100µL of TE buffer and stored at -40C before use in the further analysis.
C. Isolation of Double-Stranded Plasmid DNA 1. The bacterial cells are harvested from a 2 ml overnight culture containing cloned plasmid by transferring into a 2 ml Eppendorf tube and centrifuging at 8000 rpm for 2 minutes at room temperature.
2. The supernatant (culture media) is removed carefully and recentrifuge the pellet for 1 minute. Any traces of liquid media are removed completely from the tube.
3. The cell pellet is resuspended by vortexing briefly for 10 seconds using 100µl of Solution I. and after that kept the tube on ice.
4. 100µl of Solution II is added to the cell suspension and gently mixed by inverting the tube 10X. The tube is left at room temperature and the lysis reaction is allowed to occur for 5 minutes. A clear viscous liquid have been observed.
5. 300µl of Solution III is added and mixed by inverting the tube 10X. A white precipitate has been observed. The precipitate is pellet out by centrifuging at 10,000rpm for 5 minutes. The supernatant which contained plasmid DNA is transferred carefully into a sterile 1.5ml Eppendorf tube.
6. The plasmid DNA is precipitated by adding 800µl of cold absolute ethanol. The content is mixed gently by inverting the tube at least 10X.
7. The plasmid DNA is pellet out by centrifuging at 13000rpm for 5 minutes at room temperature. The supernatant is discarded and washed the pellet with 500µl of 70% ethanol and recentrifuged at 13000rpm for 2 minutes.
8. The supernatant is discarded as much as possible and allowed the plasmid DNA pellet to air dry for 15 minutes at room temperature. The plasmid DNA pellet is resuspended and dissolved in 50 µl of sterile ultra pure water.
D. Restriction Analysis of DNA Using Universal Restriction Endonucleases.1. The reaction for restriction digestion is prepared as the list order in the following:
Sterile ddH2O 12µl (quantity sufficient)
10 X assay buffer 2µl (one-tenth volume)
DNA (with insert) 5µl
Restriction enzyme (Pst 1) 1µl (1-10 units per µg DNA)
Total volume 20µl
2. After finished adding the above materials, we gently mixed the digestion by pipetting and incubating the reaction at the 370C for 1hours – 2 hours.
3. In the mean time, the isolated genomic DNA and plasmid DNA are ran through by using Agarose Gel Electrophoresis method 1% of agarose gel is used and electrophoresis is performed at 105 volts for 30 minutes.
4. After 2 hours, the enzyme is inactivated by heating at 650C for 10 minutes.
5. Then, the restriction digestion sample is ran through Agarose Gel Electrophoresis methods in order to get the DNA band.
E. Glass Wool Spin Column Preparation.1. A clean hypodermic needle is used to drive a hole in the end of a 650 microliter Eppendorf tubes from the inside and then the cap is cut off.
2. Gloves are worn in order to pull off a small piece of glass wool and roll it into a ball between hands. The glass wool is place in the bottom of the 650 microliter tube.1 ml pipette tip is used to pack the glass wool in the bottom of the tube.
3. The tube with the glass wool is placed into a 1.7 ml Eppendorf tube.
F. Eluting DNA from Agarose Gel Fragments.1. Ethidium bromide stained agarose gel is visualized with a transilluminator on low setting to get the fragment of interest. Then the fragment of interest is excised out with a clean razor blade in a fast pace in order to minimize the UV exposure to the DNA. After the excess liquid is removed with a tissue paper, the agarose fragment is placed into the spin column.
2. The tube at 2874 Xg is centrifuged for no more than 45 seconds to elute the DNA.
3. Transilluminator is used to check the eluent in the bottom of outer tube for the presence of ethidium bromide stained DNA.
Later, Liquid Nitrogen is added into the mortal in order to freeze the leaves... then the leaves is rapidly grind into a fine powder with a pestle and mortar as the liquids nitrogen boils off (Liquids nitrogen is added to keep the powder from thawing while grinding)
Deng La..... It is done....
Look!! Our lecture is demonstrated the technique of grinding the leaves to us...
This is the sample after CIA solution is added and centrifuge at 13000rpm for 5 minutes to obtain supernatant. The supernatant is carefully transfered into the sterile, new, clean 2.0ml microcentrifuge tube
This is the sample after cold isopropanol is added and centrifuge at 13000 rpm for 2 minutes in order to precipitate nucleic acids. Then after that it is store at -20 degree celcius freezer for at least 30 minutes.
