Chloroplasts are unique organelles in plant cells and contain DNA. chloroplast DNA is in the form of a ring, and the chloroplast DNA (cpDNA) of higher plants varies in size from 120 to 160 kilobases (kb), with the largest chloroplast DNA found now reaching 217 kb.
Operation method
basic program
Principle
In this experiment, fresh tissue homogenate was prepared and filtered to isolate intact chloroplasts, which were then separated from other subcellular structures by sucrose density gradient centrifugation. The intact chloroplasts were digested by proteinase K and then extracted by phenol-chloroform to obtain high-purity chloroplast DNA.
Materials and Instruments
Fresh young leaves of plants Move All of the following operations were performed at 4°C unless otherwise indicated. Pre-cool the stirrer and centrifuge tubes, use refrigerated buffer and operate in an ice cylinder. 1. Prepare 750 ml of buffer A and add BSA to 500 ml of buffer before use. 2. 25-30g of healthy young leaves were collected and the plants could be incubated in the dark for 24-28h prior to extraction to reduce the starch content. If the plant material has been infected or soiled, it can be treated with sodium hypochlorite (5%) for 5 min and then rinsed with tap water for 2 to 3 times. 3. Remove the veins in the middle of the leaves and weigh them. This method is applicable to about 20g of plant material. The leaves were cut into 1cm2 pieces, and 10g of the pieces were placed in about 200-250ml of buffer A containing BSA (BSA was added only to the buffer A used for homogenization), and homogenized in a mixer at high speed for about 10s each time, and then repeated 2 or 3 times. 4. The extract was filtered through a 50-μm nylon sieve. The remaining fragments were homogenized and filtered again in 200-250 ml of buffer A containing BSA. The homogenized material that could not pass through the 50 μm nylon sieve could be pooled and homogenized again for 10 s each time, repeated twice, and then filtered. 5. The extract was filtered for the second time using a 20 μm nylon sieve. 6. The extract was centrifuged at 3000r/min for 10min. 7. The precipitate was gently resuspended in 30 ml of BSA-free buffer A with a soft brush and then centrifuged. The white spots on the bottom of the green precipitate are starch, and resuspension of starch should be avoided as much as possible. Depending on the amount of starch, this rinsing step can be repeated 4 to 6 times. 8. While rinsing, a sucrose gradient can be prepared. The bottom layer of the gradient is 3.5 ml of 60% sucrose solution, the middle layer is 3.5 ml of 40% sucrose solution, and the top layer is 3.0 ml of 20% sucrose solution, and the different gradient layers should be mixed gently to obtain the diffusion intermediate phase. A long pasteurized pipette can be used to carefully stir up and down the layers a few times (the opening at the end of the pasteurized pipette should be closed). 9. Carefully resuspend the precipitate in a total volume of 2-6 ml of buffer A. Add the suspension to 2-6 tubes with graded gradients. The gradient was equilibrated and then centrifuged at 26000r/min 4°C for 1h on a horizontal rotary head. 10. The chloroplast bands located in the middle layer of 45%-20% sucrose solution were transferred to a 50 ml centrifuge tube using a wide mouth pipette. 11. Slowly add 3 times the volume of buffer A (to prevent chloroplast rupture). Add buffer A drop by drop at the beginning and stir gently (it takes about 10-15 min to add all the buffer). 5000r/min centrifugation for 5min, collect the chloroplasts. 12. 12. Carefully resuspend the precipitate in 3 ml of buffer A, add 1/5 volume of buffer B (add proteinase K before use), and hold at 50 ℃ for 15 min to make the chloroplasts cleaved. 13. Extract chloroplast DNA twice at room temperature. Add 1 times the volume of phenol (saturated with TE), invert the tube several times to mix. The two phases were separated by centrifugation at 5000 r/min for 10 min at room temperature. Collect the upper layer of the solution, and then extract it once with 1x volume of phenol-chloroform (1:1, V/V). 14. Transfer the DNA solution (aqueous phase) to a 30 ml centrifuge tube, add 1/10 volume of 3 mol/L sodium acetate and 2.5 times the volume of 99% ethanol, and precipitate the DNA at -20℃ overnight. 15. Centrifuge the sample at 10,000 r/min for 15 min at 4℃ and collect the DNA. 16. Discard the supernatant and vacuum-dry the precipitate (do not over-dry the precipitate). 17. Dissolve the DNA in 400 μl of TE buffer, which may take several hours, and place the tubes on ice. 18. Transfer the DNA to an Eppendorf tube, add 1/10 volume of 3 mol/L sodium acetate and 2 volumes of 99% ethanol, and precipitate the DNA at -20 overnight. 19. Collect the DNA by centrifugation at 13000 r/min for 15 min at 4℃ in a microcentrifuge. 20. Discard the supernatant, wash the precipitate with 20% ethanol, centrifuge again and repeat the washing step. Centrifuge and vacuum dry the precipitate. 21.Dissolve the precipitate in 50~150μl of TE buffer and store at -20℃ or 4℃.The DNA yield can be 10~100μg. Caveat 1. The amount of chloroplasts added to each tube of sucrose gradient will affect the purity of the DNA. In general, 10-20 g of chloroplasts obtained from fresh leaves are added to 25 ml of sucrose gradient. The gradient solution used in this experiment is 10 ml, in order to avoid overloading the gradient solution with a smaller amount used in this procedure, the chloroplasts can be divided into 2-6 tubes, and the total volume of the gradient solution should be determined according to the feel of different plant materials. 2. People usually use CsCl gradient solution to further purify chloroplast DNA, and CsCl purified chloroplast DNA can be used for cloning, but CsCl gradient centrifugation is more time-consuming. This experimental procedure also yields highly purified chloroplast DNA, which can be tested for cloning. Common Problems Specific reagent description: 1. Buffer A (extraction buffer): 0.3 mol/L sorbitol ,50mmol/L Tris-HCl, 20mmol/L EDTA, 0.1% BSA (added before use). 2. Buffer B (lysis buffer): 0.5% SDS, 50mmol/L Tris-HCl, 0.4mmol/L EDTA, 0.1% Proteinase K (W/V), pH 8.0. Add Proteinase K before use, and heat up the buffer before use, because the buffer B will be precipitated at room temperature. 3. TE buffer: 10mmol/L Tris, 5mmol/L Na2EDTA, pH 7.5. 4. Sucrose gradient: 60%, 45% and 30% sucrose, dissolved in Buffer A. 5. Nylon sieves (50 μm and 20 μm), phenol (TE buffer saturated, pH 8.0), 99% and 70% ethanol, 3 mol/L sodium acetate. For more product details, please visit Aladdin Scientific website.
Buffer A (extraction buffer) Buffer B (lysis buffer) TE buffer Sucrose solution Sodium acetate Ethanol
Stirrer Freezing centrifuge (Sorvall, Beckman, etc.) Brushes (good quality art brushes) Ultracentrifuge with flattened rotor head and suitable tubes Thermostatic water bath 50 ml centrifuge tubes for phenol extraction 30 ml centrifuge tubes 1.5 ml microcentrifuge tubes Microcentrifuge (1.5 ml tubes) -20°C Refrigerator Vacuum dryer