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0. What causes the proteins to move through the SDS Acrylamide gel when the electric current is turned on.

1. We want to make 1 Liter of 1x running buffer from the 10x stock. You will add _(a) mL of 10x stock to _(b) mL water.

2. In SDS PAGE, we will denature the proteins before we load them on the gel. What are the two ways by which we will denature the proteins in the experiment?

3. Why do we need to denature the proteins before loading them on the gel?

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Bunny Greenfelder
Bunny GreenfelderLv2
28 Sep 2019
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Related questions

10. When you make your agarose gel, you will need to make 50 ml of 1% (w/v) agarose in 1X TAE. How much agarose will you require? [you will make this later, not on Solution day] When adding your DNA samples to the agarose gel, you will need to add loading dye to the samples. Loading dye contains: • glycerol, which helps the samples sink into the sample wells of the gel; and • tracking dyes so that you have a way of estimating how far the samples have travelled through the gel during electrophoresis. The recipe for 6X loading dye is as follows: • 0.25% bromophenol blue (w/v) • 0.25% xylene cyanol (w/v) • 30% glycerol in water (v/v)

11. Provide a recipe for 10 ml of 10X loading dye, using the information above. (Important tip: Remember that you have already made 30ml of a 50% stock solution of glycerol. I recommend determining the concentration of glycerol required for 10X loading dye, then figuring out how much of your 50% glycerol stock will be needed to make 10ml of 10X loading dye.)

Solution to make

Exact pH of final solution (if appropriate)

Final container for solution

Completed (Y or N)

100 ml of 0.9% (w/v) NaCl

100 ml of 1.0M Tris, pH 7.6

40 ml of 50X TAE

Assignment: For each question, describe how you would make the solution using the stocks or reagents listed below. On our lab prep shelves, we have the following solutions and powders:

Sodium citrate (FW = 43.7 g/mol)

SDS powder (FW = 288.38 g/mol)

0.5 M EDTA IPTG powder (FW = 238.3 g/mol)

20 mg/mL X-gal solution

1000X ONPG

Premade LB

Premade LB agar

0.5 mM Colchicine

50X gel running buffer

Agarose powder

95% ethanol

100 mg/mL Ampicillin

10X BamHI restriction enzyme digestion buffer

milliQ water dimethylformamide (DMF)

Example 1. (DNA isolation lab) How would you make 100mL of 0.15M Sodium Citrate, 5% SDS, 0.001M EDTA solution (one solution that contains all of these reagents)? Answers 0.65 g Sodium citrate 5 g SDS 0.2 mL of 0.5 M EDTA Add 75 ml of water and mix well. Then add water up to 100 ml to complete.

Example 2. (B-gal) How would you make a 10 fold dilution of 20 mg/ml X-gal? X-gal is not soluble or stable in water, but it is in dimethylformamide. What would the concentration of the diluted solution be? Answer Add 1 ml of the stock solution to 9 ml of DMF. 2 mg/ml

Problem for you

1. How would you make 500 mL LB = 40ug/mL X-Gal? ( the LB is made, so you just need to figure out how much X-Gal to add)

How would you make 500mL LB = 0.1mM IPTG? You cannot add solid IPTG to media. Instead use a sterile 1M stock of IPTG

How would you make 500mL LB + 40ug/mL X-gal + 0.1mM IPTG?

Please show all work that got you to your solution

thank you!

I need a good introduction and conlcusion of this experiment below is the expermeint and my results.

In this experiment you will observe the action of the enzyme amylase on starch. Amylase changes starch into a simpler form: the sugar maltose, which is soluble in water. Amylase is present in our saliva, and begins to act on the starch in our food while still in the mouth.

Exposure to heat or extreme pH (acid or base) will denature proteins. Enzymes, including amylase, are proteins. If denatured, an enzyme can no longer act as a catalyst for the reaction.

Benedict's solution is a test reagent that reacts positively with simple reducing sugars like maltose, but will not react with starch. A positive test is observed as the formation of a brownish-red cuprous oxide precipitate. A weaker positive test will be yellow to orange

PRE-LAB:

Add 1g of cornstarch to a beaker containing 100ml of cold distilled water. While stirring frequently, heat the mixture just until it begins to boil. Allow to cool.

PROCEDURE:

1. Fill the 250-mL beaker about 3/4 full of water and place on the hot plate for a boiling water bath. Keep the water JUST AT BOILING.

2. Mark 3 test tubes A, B and C. "Spit" between 1 and 2 mL of saliva into each test tube.

3. Into tube A, add 2 mL of vinegar. Into tubes B and C, add 2 mL of distilled water. Thump the tubes to mix.

4. Place tube B into the boiling water bath for 5 minutes. After the five minutes, remove from the bath, and place back into the test tube rack.

5. Add 5 mL of the starch solution to each tube and thump to mix. Allow the tubes to sit for 10 minutes, occasionally thumping the tubes to mix.

6. Add 5 mL of Benedict's solution to each tube and thump to mix. Place the tubes in the hot water bath. The reaction takes several minutes to begin.

OBSERVATIONS:

Tube A: Starch + saliva treated with vinegar (acid)

Was the test positive or negative? Negative

What does this indicate? The vinegar stops the action of amylase from catalyzing the starch therefore the solution becomes cloudier. This indicates that vinegar (acids) distort the shape of proteins in the salivary amylase.

Results: The solution turns cloudy.

Conclusion: Acids denature the shape of proteins in the salivary


Tube B: Starch + saliva and water, treated in a boiling water bath

Was the test positive or negative? Negative

What does this indicate? It indicates that when enzymes are exposed to extreme temperature for some time they are denatured and therefore become inactive.

Results: The benedict’s solution retained its color as the reaction did not occur. Extreme temperatures denatures enzymes, therefore when the salivary amylase was introduced to a hot water bath for five minutes the amylase was denture.

Conclusion: Salivary amylase/enzymes when introduced to hot temperatures it is a denatured therefore does not react on the substrate.


Tube C: Starch + saliva

Was the test positive or negative? Positive

What does this indicate? This indicates that the salivary amylase successfully worked on the starch.

Results: The benedict’s solution turned brown as an indication of the presence of sugar.

Conclusions: Salivary amylase acts on starch in favorable conditions, ie is optimum te

Please provide a lab report that explains your procedure, results and conclusions. Remember to include pictures of the lab set-up and results. Also, I want you to expand this initial experiment by proposing and testing (in the form of an additional experiment) a testable hypothesis that involves the above general concept. You will be graded on your use of the scientific method and the creativity/complexity of your experiment. Both the above experimentation and your original experiment may be included together into one experiment

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