Isolation of Chloroplasts from Spinach Leaves by fractionation

Theory

  

Chloroplasts are organelles 5-10 um in size in plants which perform the function of photosynthesis.  These structures are mainly located in the palisade parenchyma of the mesophyll cells in the leaf. Chloroplasts belong to a family of specialized organelle called the plastid. The stem, leaves and unripened fruit in all the plants contain chloroplast. But leaves are major part of plants performing the process of photosynthesis. The green color of these structures in plants is due to the presence of a pigment called chlorophyll which resides in the chloroplasts. 

Chloroplasts are found in the mesophyll cells of the leaves. The chloroplast is divided into three compartments bounded by three membrane systems: an intermembrane space between the inner and outer membranes, the stroma and the thylakoid lumen.  Chloroplasts have a double membrane structure called the chloroplast envelop.  The chloroplast envelop has an inner membrane and an outer membrane. A third membrane system called the thylakoid membrane surrounds the thylakoids in the stroma.  

Chloroplasts are located inside the thylakoid membranes.  Thylakoid membrane consists of the thylakoids which are flattened discs arranged on top of the other and they are termed as grana.  The thylakoids are located inside the stroma. Photosynthesis takes place in the thylakoid membranes. The chlorophyll molecules absorb light in the form of photons and this leads to the emission of electrons by the chlorophyll molecules. This drives the hydrogen ions across the membrane surrounding the thylakoid stack. This leads to the formation of an electrochemical gradient which drives the production of ATP. 

Principle

The cells of spinach leaves are disrupted mechanically by using a blender or homogenizer, freeing the untethered organelles, which can then be sorted out from each other by filtration and differential centrifugation as cell fractions. Filtration will remove large debris (e.g., cell walls) and unbroken cells, providing a filtrate that contains organelles (nuclei, chloroplasts, mitochondria, ribosomes), small membrane vesicles, and soluble components; most of these will not be visible in the light microscope. Low-speed centrifugation will sediment remaining large bodies from the filtrate, and moderate-speed centrifugation will sediment chloroplasts, leaving mitochondria and ribosomes and soluble components in the supernatant. (The mitochondrial fraction could be collected by high-speed, and ribosomal and membrane vesicle fractions by ultra-high-speed centrifugation.) Repeated rounds of differential centrifugation can be used to further purify the chloroplasts when highly purified preparations are required.

Procedure:

Keep the leaf and all fractions ice cold throughout steps 1 - 6 and 10.  

1. Take 8 grams of de-veined leaf tissue rinsed in ice water, blotted and cut into pieces about 1 cm square.
2.  Place the leaf pieces in a pre-chilled blender cup containing 40 ml of ice-cold 0.5 M sucrose. 
3. Blend for 15 sec. at top speed, pause about 10 sec., then blend again for 10 sec. 3.
4. Remove the ice from the 100-ml beaker, then squeeze the leaf homogenate through four layers of pre-chilled cheesecloth into the cold beaker by twisting the top corners of the cloth around each other.
5. Pour 14 ml of the homogenate into each of two centrifuge tubes and centrifuge at 200g for 5 min.
6. Using a Pasteur pipet, transfer each supernatant (containing the chloroplasts) to a second centrifuge tube and centrifuge at 1000g for 7 min.
7.  Using the pipet, discard the supernatant but be careful not to disturb the pellet.
8. Pour 2 ml of phosphate buffer onto the pellet and gently suspend it by moving it up and down in the pipe.
9. Using a clean Pasteur pipet, add buffer until you have a total volume of 8 ml and mix the diluted suspension using the pipet. This is the chloroplast suspension.
10. Examine under the microscope.
11. Using a hemacytometer, determine the number of chloroplasts per mL of suspension media or observe mitochondria under microscope.

Estimation of chlorophyll a concentration of the suspension.

1. Take 4.75 ml of 80% acetone in tube tube.
2. Add 0.25 ml of chloroplast suspension, mix well, and read the Centrifuge at 3000xg for 2 minutes.
3. Take 1000ul of the supernatant and transfer into a cuvette and measure the absorbance at 650 nm. Use 80% acetone as blank.
4. Take the average of the two values and estimate the mg/ml chlorophyll concentration using the following formula:

A at 650 x df /36 = mg/ml chlorophyll.  

Where A 650 is the absorbance at 650 nm and 36 is the extinction coefficient of chlorophyll.

References:

Joly D and Carpentier R (2011). Rapid isolation of intact chloroplasts from spinach leaves. Methods Mol Biol. 684:321-325.

Tetsuko Takabe, Mikio Nishimtjra and Takashi Akazawa (1979). Isolation of Intact Chloroplasts from Spinach Leaf by Centrifugation in Gradients of the Modified Silica “Percoll”. Agricultural and Biological Chemistry, 43(10): 2137-2142.

Extraction of Plasmid DNA from bacteria (Alkaline lysis method)

Principle:

The bacterial cells are lysed with lysozyme and SDS at high pH and the lysate is then neutralized. The plasmid undergoes renaturation but not the chromosomal DNA. The later gets precipitated out in the form of protein-DNA-SDS complex. Subsequent deproteinization with phenol: chloroform, plasmid DNA is precipitated with ethanol by spinning at high speed. 

Requirements:

1. Overnight Log- phase culture in LB broth (10 g tryptone, 5 g yeast extract and 10 g NaCl, 1L DW)
2. Solution I (Lysis buffer): 25 mM Tris, 50 mM Glucose, Lysozyme (GNB: 0.5 mg/ml, GPB: 3-5 mg/ml), pH 8.0
3. Solution II (Lysis): 0.2M NaOH, 1% SDS 
4. Solution III: 3 M Sodium acetate, pH 4.8 by acetic acid
5. Phenol: Chloroform (1:1)
6. RNase A: (1 mg/ml in 5 mM Tris-HC1, pH 8.0)
7. Absolute ethanol/Isopropanol
8. 70% ethanol
9. TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0; Autoclave before use
10. Water bath
11. Cooling centrifuge (upto 20000 rpm)
12. Micropipettes (1 – 10, 10 -100, 100 – 1000 µl ) and sterile tips

 Procedure: 

1. Take 1.5 ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000 rpm for 2 min.
2. Remove the supernatant and spin once with same volume as above to collect more cell mass.
3. Remove the supernatant.
4. Add 100 µl Sol. I. Re-suspend and keep for 30 min at RT.
5. Add 200 µl freshly prepared Sol. II and mix gently (Do not vortex). Keep for 5 min at 4°C.
6. Add 150 µl ice cold Sol. III and mix gently. Keep in ice bath for 10 min.
7. Spin at 10000 rpm for 5 min at 4°C. Collect Sup-T into new MFT.
8. Add 1-2 µl of RNase and incubate for 10 min at RT.
9. Add equal vol. of Phenol:Chloroform (1:1), mix gently and keep for 10 min at RT.
10. Centrifuge at 8000 rpm for 10 min at 4ºC and collect the supernatant in a new sterile MFT.
11. Add two vol. of absolute ethanol and stand it for 10 min in cold.
12. Spin at 13000 rpm at 4ºC for 10 min.
13. Remove the Sup-T into new MFT and add 1000 µl of 70% ethanol. Mix well and keep at 4°C for 30 min.
14. Spin at 13000 rpm at 4ºC for 10 min. Remove Sup-T.
15. Dissolve the pellet collected during spin in 50 µl TE and store in deep freeze.

Solid-State Drive (SSD)

An SSD (solid-state drive) is a type of nonvolatile storage media that stores persistent data on solid-state flash memory and has no moving parts unlike a hard disk drive (HDD), which stores data on a spinning disk. Two key components make up an SSD: a flash controller and NAND flash memory chips. The architectural configuration of the SSD controller is optimized to deliver high read and write performance for both sequential and random data requests. SSDs are sometimes referred to as flash drives or solid-state disks.

 To prevent volatility, SSD manufacturers design the devices with floating gate transistors (FGRs) to hold the electrical charge. This allows an SSD to retain stored data even when it is not connected to a power source. Each FGR contains a single bit of data, designated either as a 1 for a charged cell or a 0 if the cell has no electrical charge.

Unlike a hard disk drive (HDD), an SSD has no moving parts to break or spin up or down. A traditional HDD consists of a spinning disk with a read/write head on a mechanical arm called an actuator. The HDD mechanism and hard disk are packaged as an integrated unit. Businesses and computer manufacturers have used spinning disk historically, owing to their lower unit cost and higher average durability, although SSDs are now common in desktop and laptop PCs.

Things Should Know before you Buy SSD

1. SSD Disk Capacity

When you buy SSD for your laptop or Desktop, you can keep your SSD for OS and Apps and secondary HDD for your data in a Dual Drive Configuration. A 40GB SSD will be enough to run your PC on Windows or MAC operating system with a couple of essential apps. But if you can afford 80GB SSD, that will be a decent size and no need to worry about low memory for a while.

You can’t go with a smaller size if you are planning to replace your entire HDD with new SSD (Single Drive Configuration). An SSD with 250GB should be the starting point for your computer to take care of your OS, Application, and Data. There are SSDs that comes in Terabytes if you are ready to burn some additional bucks.

2. SSD Performance

Manufacturers are specifying the SSD performance related to the Sequential Read, and Sequential Write speed that typically goes up to 500MB/s in reading and bit lower in write.

3. SSD Data Random Transfer Rate:

SSD Random Write Speed and Random Read Speed is another benchmark to measure the performance of SSD. Random Read/Write Testing performs with small blocks of at random locations on the drive. Naturally, this process will be slow compared to Sequential Data handling. Typically, around 25% read-write operation will be random for an actual user.

4. SSD Flash Memory:

Solid state drives are based on flash memories with different level of NAND memories. The table below will give us a quick review of technologies those are available in the market now.

5. SSD Endurance MTBF (Reliability):

Mean Time Between Failures (MTBF) is the manufacturer’s estimate of total running hours of product shipped divided by the number of failed units. Long MTBF is always useful indication but no guarantee that the product can last that long. The reliability for SSDs falls in the range of a couple of million hours.

6. SSD Hardware Interface:

Most of the SSDs are coming with built-in Serial ATA (SATA) interface SATA support. The transfer speed can vary on the SATA versions. The new SSDs support up to SATA III that offers 6GB transfer speed where SATA II is capable of transferring 3GB where SATA I is limited to 1.5GB in data transfer.

Erythrocyte Count by haemocytometry

Erythrocyte Count by haemocytometry

Principle

The blood specimen is diluted (usually 200 times) with red cell diluting fluid which does not remove the white cells but allows the red cell to be counted under high power (400 X) magnification in a known volume of fluid. Finally, the number of cells in undiluted blood is calculated and reported as the number of red cells/µl of whole blood. The whole blood is held inside a micro chamber made by a slide with a grid at the bottom. The number of cells counted in the volume of fluid held in the chamber.

Reagent/ Requirements:

1. Sample: Whole blood (anti coagulated venous blood).

2. Diluting Fluids: Red cell diluting fluid- isotonic to red cells which prevent hemolysis.

              Trisodium citrate                                                        3.13 gm

             Commercial formaldehyde (37% formalin)                1.0 ml

             Distilled water                                                            100 ml

3. Blood sahli pipette 20 µl and 50 µl.

4. Hand tally counter

5. Hemacytometer or counting chamber (Neubauer chamber).

6. Microscope.

7. Test tubes.

Procedure:

1. Take a 25 ml flask or test tube of 20 ml and labelled with sample code.
2. Mixed the sample well (at least for 1 minute by gentle shaking; be care full of lysis of RBC) and dilute the uncoagulated sample (usually to 200 times or as per requirements) with diluting fluid. 200 times dilution can be done by mixing 0.02 ml blood sample with 4 ml diluent (exactly 3.98 ml).
3. Mix the diluted blood sample very well with help of pipette.
4. Take a haemocytometer (clean and dry) and place on the flat surface. Place the coverslip on the counting chamber.
5. Take small amount (about 20 µl) of well mixed blood sample (before this for this fill the pipette and dispense on same flask for 3 times- rinsing).
6. Allow a small drop of diluted blood hanging from the pipette and to seep into the counting chamber by capillary action. Make sure that there is no air bubble and there is no overfilling beyond the ruled area. (If the liquid overflow into the channel between the two chambers, repeat from no.4.
7. Leave the counting chamber on the flat surface for 3 minutes to allow the cells to settle.
8. Observe the slide under 10X and locate the large square in the center with 25 small squares. Examine uniform distribution of RBCs.
9. Switch to 40X (high power) and focus on smaller upper left corner which is divided into 16 smaller squares (3 lines boundary on each sides).
10. Count the number of RBCs on the small squares (0.2 X 0.2 = 0.04 sq. cm) falling within the square and those touching the outside lines on the top and inside margin on the left (discarding those that touch the inside margin at the bottom and free margin on the right).
11. Repeat the counting with four other squares (upper right corner square, lower right corner square, lower left corner square and center square divided into smaller 16 divisions in written order or those marked r as in figures.
12. Make the total of count.

                          

Reference: Mukherjee KL (2008). Medical laboratory Technology- A procedure manual for routine diagnostic test. Volume 1. Tata McGraw-Hill publishing Company limited, New delhi.

Agarose gel electrophoresis of DNA

Principle:

Agarose gels are more porous and have a larger pore size as compared to polyacrylamide gels and, therefore, used to fractionate larger macromolecules such as DNA or RNA. Porosity of a gel is determined by concentration of agarose – higher the agarose concentration, smaller the pore size and vice versa. When an electric field is applied across the gel, DNA molecules that are negatively charged at neutral pH, migrate towards oppositely charged electrode at rates determined by their molecular size and confirmation. Since charge to mass ratio of NA is constant, the rate of migration is inversely proportional to log₁₀MW, i.e., smaller DNA molecules will travel faster as compared to the larger ones. Further, DNA molecules of the same size but with different conformations travel at different rates. The order of the migration velocity in the increasing order of various forms of DNA is: Supercoiled DNA > linear double stranded DNA > open circular DNA.

Table 1: Percentage of gels and range of resolution for DNA electrophoresis

% Agarose	Range of resolution (linear dsDNA, Kbp)	% Acrylamide	Range of    resolution
			dsDNA (bp)	ssDNA (nt)
0.5	30 – 1.0	3.5	100 – 1000	750 – 2000
0.7	12 – 0.8	5.0	75 – 500	200 – 1000
1.0	10 – 0.5	8.0	50 – 400	50 - 400
1.2	7 – 0.4	12.0	35 – 250
1.5	3 – 0.2	15.0	20 – 150
		20.0	5 - 100

Requirements:

1. 1 X TAE buffer at pH 8.0 (50 X, 24.2 gm Tris, 5.71 ml GAA, 11.1 ml of 0.5 M EDTA, 100 ml DW) Autoclave before use
2. Agarose gel in 1 X TAE
3. Loading dye  (6 X, 6.0 ml of 50% Glycerol, 1.0 ml of 2% BPB, 3 ml sterile DW)
4. Ethidiun Bromide (EtBr)*: 10 mg/ml stock
5. λ/HindIII Marker (23.13, 9.42, 6.56, 2.32, 2.07, 0.56 and 0.13 Kb)
6. Plasmid DNA (pUC18, pBR322)

Procedure:

1.  Prepare 0.8% agarose gel in TAE buffer.

2.  Dissolve agarose completely in micro-oven and cool to 60°C.

3. CAREFULLY, add EtBr into the gel solution to final concentration of 0.5 µg/ml.

4. Pour the molten gel into gel-mould. Immediately position a comb in the mould.

5.  Let the gel cool for 30 minutes.

6.  Pour TAE buffer into the gel buffer reservoirs.

7.  Prepare sample taking 20 µl of DNA sample and mix with 4µl blue juice (6 X).

8.  Carefully remove the comb.

9.  Load the DNA (15 - 20 µl) per well, flanking wells with similarly processed DNA size standard.

Note: the amount of the sample that can be loaded in a well depends on the thickness of the gel as well as dimensions and placing of the comb.

10. Put the lid on the gel apparatus, attach the electrodes and adjust voltage to 100 volts.

11. Allow the gel to run until line of blue juice is visible       near the end of the gel.

12. Turn off the current and visualize the gel in UV - Tran illuminator.

13. Interpret the results.

*Note: EtBr is carcinogen, so, handle with gloves.

Quantitation of Carbohydrates by Anthrone method.

Quantitation of Carbohydrates by Anthrone method.                                    
                                                                                                                  Khadga Bikram Angbuhang
                                                                                                         GoldenGate International College

Principle:

Anthrone reaction is the basis of a rapid and convenient methods for the determination of hexose, aldopentoses either in free form or in when present as polysaccharide. The reaction is not suitable if protein containing large amount of tryptophan is present, since a red color is obtained under these conditions.

In this assay, carbohydrates are dehydrated by using conc. H₂SO₄ to form furfural and its derivatives, which in turn condenses with anthrone to form a bluish green complex with an absorption maximum at 620 nm.

D- glucose + conc. H2SO4                              Hydroxymethyl furfural +3H2O

Hydroxymethyl furfural + Anthrone                              Bluish green complex

Materials and reagents:

1.  Anthrone reagents
2.  Conc. H₂SO₄ 
3. Glucose standard 50 μg/ml
4. Sample carbohydrates (Carbohydrates) 

             Procedure:

1. Take 3 test tubes and label as Test, Standard and Blank.
2.  Add 1 ml Glucose sample in test labelled test tube, 1 ml glucose standard to standard test tube and 1 ml D/W (protein free) to blank.
3. Add 4 ml of anthrone reagent to each tubes.
4. Place all tubes in a boiling water bath for 10 minutes.
5. Cool and read the absorbance at 620 nm against the blank.

Absorbance of sample X concentration of standard calculate the concentration as:

 Concentration glucose (μg/ml):              Absorbance of Standard.