SDS – PAGE (10%)
PREPARATION OF GELS:
RESOLVING GEL PREPARATION:
1. Pipette out 2.66 ml of 30% Acrylamide and pour in beaker.
2. Add 2 ml of Lower Tris (L-Tris, 1.5 M, 8.8 pH) to this.
3. Take 1.96 ml distilled H2O and pour it into this beaker.
4. Now take 1.2 ml Glycerol and add to this mix.
5. Pipette out 80 microliter of 10% SDS (Sodium Dodecyl Sulfate) and pour into the beaker.
6. Add 80 microliter APS (Ammonium per sulfate) as well as 8 microliter TEMED to this as well.
7. Pipette out 4 microliter of TEMED and add it to the beaker as well.
8. Mix these contents together properly and resolving gel (lower gel) is prepared.
CASTING GEL PREPARATION: (3 ml)
1. Pipette out 0.3 ml of 30% Acrylamide and pour in beaker.
2. Add 0.75 ml Upper Tris
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After polymerization of lower gel, now add the Casting Gel and keep it undisturbed for around an hour, so that it can solidify.
After this, sample preparation is done. Add sample buffer to samples in 3:1 ratio. The Sample Buffer is made of SDS, BPB and Beta Mercaptoethanol.
(SDS denatures the proteins, BPB is used as a tracking dye while Beta Mercaptoethanol breaks sulfide bonds.)
Heat the samples at 80 degrees Celsius for 5 minutes in a water bath. This helps in protein denaturation.
Put the cassette into the buffer dam and fill it with the running buffer. For SDS-PAGE, running buffer is prepared by taking 40 ml Tris-Glycine and adding 4 ml of 10% SDS to it. Its volume is raised up to 400 ml by adding distilled water.
Load these samples into wells created in the gel.
Run the electrophoresis unit at 5 mA and 20 V for entry of sample into lower gel.
Once the samples enter the lower gel, alter the current to 10 mA and set the voltage at 90-100 V.
Leave this setup undisturbed for around 3 hours.
Once the gel has run and the samples have reached the bottom end of the cassette (a faint blue line is visible), the gel can be taken
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Only a limited sample analysis can be done using electrophoresis as this technique is quite specific in nature. Therefore, at a time, electrophoresis can only be used for analyzing samples obtained from some particular or specific tissues, and from nowhere else. This specificity results in accurate results, but can be detrimental in cases where researchers are interested in more widespread studies or results.
3. Measurements obtained through electrophoresis are not always precise. Though gel electrophoresis is very effective in separating similar proteins with different molecular weights, and nowadays it uses 2 dimensional techniques to obtain further precision, most measurements made by this technique can be considered semi-quantitative at best. Even today, if obtainment of precise mass (weight) of proteins is desired, mass spectroscopy must be carried out after the protein has been purified by electrophoresis.
Furthermore, random errors are quite rampant and samples are usually run multiple times to get clean and correct
Each subsequent trial will use one gram more. 2.Put baking soda into reaction vessel. 3.Measure 40 mL vinegar. 4.Completely fill 1000 mL graduated cylinder with water.
Then titrate with the sodium thiosulfate solution as in the standardization procedure, adding 6 drops of starch indicator near the end of the titration. Record the volume of thiosulfate solution used in the titration. Make a duplicate
We used the pipette filler and filled the glucose rinsed pipette to add 10ml of 10% of glucose in test tube 0.
Now, assemble and arrange all of the needed supplies so that they are easily accessible. Connect the IV tubing to the solution bag and allow the fluid in the bag to run through the entire length of the tubing, also known as priming the tubing. When this is done, clamp the tubing closed. You will then need to tear several pieces of tape, six to eight inche...
Once the mixture had been completely dissolved, the solution was transferred to a separatory funnel. The solution was then extracted twice using 5.0 mL of 1 M
In a laboratory scientist will use a process called gel electrophoresis to separate DNA fragments. The DNA is cut into different sized fragments as a result from using restriction enzymes. The different sized DNA fragments are organized injected on agarose gel with an added substance that helps it glow after the test. DNA is negatively charged. Electricity is producing a positively charged are and a negatively charged area. Opposites attract and as a result the negatively charged DNA will move quickly to the positively charged area. Smaller DNA fragments will run faster the larger DNA fragments. After the electricity is turned off smaller DNA fragments will be closer to the positively charged area and the larger DNA fragments will be farther from the positively charged area. While it is glowing scientist can take a picture of the data and record the results and compare DNA samples to look for any abnormalities.
I am going to use a range of concentrations to enable me to get a good
3.) Divide your 30g of white substance into the 4 test tubes evenly. You should put 7.5g into each test tube along with the water.
In a 100ml beaker place 50mls of water, measure the temperature of the water and record this initial temperature onto a table. Set the timer and add one teaspoon of Ammonium Nitrate to the water, stir this continuously until the Ammonium Nitrate has dissolved.
== § Test tubes X 11 § 0.10 molar dm -3 Copper (II) Sulphate solution § distilled water § egg albumen from 3 eggs. § Syringe X 12 § colorimeter § tripod § 100ml beaker § Bunsen burner § test tube holder § safety glasses § gloves § test tube pen § test tube method = == = =
About 25ml of distilled water was added. Then 2-3 drops of phenolphthalein indicator solution were put in. It was swirl to dissolved the solid.
2. In the large beaker, put water and boil it completely. After that, remove the beaker from heat. 3. Sample tubes (A-D) should be labeled and capped tightly.
Gel electrophoresis is used in a variety of settings, particularly in molecular biology. Besides being used to separate nucleic acids, such as DNA and RNA, gel electrophoresis is also employed to divide proteins (Gel Electrophoresis). According to research, electrophoresis is applied for the following reasons, "To get a DNA fingerprint for forensic pur...
of distilled water. For the 1M solution I added 50 cm3 of HCl and 50
Through the advancement of the analytical techniques we can move into the regime where the details play a major role in understanding life sciences, for example in the field of metabolomics. It involves the determination of a small set of molecules known as metabolites with the molecular weights typically in the range lesser than 2500Da in organisms or cells. In contrast to genomics involving the combinations of gene or protein alphabet, it is structurally far more diverse. Also it is required that they must be determined at native concentrations as there are no general amplification protocols for metabolites existing yet. Also there is currently no method which provides this branch with the throughput and sensitivity of genomics.