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27 Jun 2019

Please show how the following would be prepared?

Pipette 1 Âµg of total RNA into a 1.7 ml RNAse-free microfuge tube. Add the appropriate amount of loading buffer plus SYBR Green; the stock solution is 6X (final concentration should be 1X). If needed, adjust volume with DEPC-treated water so that the final volume is 10 Âµl

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Jarrod RobelLv2

28 Jun 2019

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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: â¢ glycerol, which helps the samples sink into the sample wells of the gel; and â¢ 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: â¢ 0.25% bromophenol blue (w/v) â¢ 0.25% xylene cyanol (w/v) â¢ 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

After your frustration with tissue culture, you finally get your cells passaged and decide to set up your cDNA synthesis reaction, PCR, and agarose gel. You have extracted RNA from your cells, and now you need to proceed with the cDNA synthesis.

The first step is to determine the concentration of your RNA. You dilute your RNA 1:250, vortex it, move it to the cuvette, and run it on the spectrophotometer. The spec tells you that your concentration of RNA is 22Âµg/mL with an A260 of 0.32 and an A280 of 0.2. What is your ACTUAL RNA concentration, and what is its purity?

After determining your actual concentration of RNA, you are ready to make your cDNA. The protocol calls for the following for a total of 20ÂµL:

5x iScript Reaction Mix â 4ÂµL

iScript reverse transcriptase â 1ÂµL

Nuclease free water â x ÂµL

RNA template (100fg to 1ug total RNA) â x ÂµL

You decide to use 500ng (or 0.5Âµg) of RNA in this reaction. How many ÂµL of your RNA do you need, and how much water will you put in the tube?

You make the cDNA and get ready to set up your PCR for CD-10. The Qiagen protocol is as follows to a total of 50ÂµL:

cDNA â 2ÂµL

MgCl (25mM) â 4ÂµL

CD-10 Forward Primer (10nM) â 1ÂµL

CD-10 Reverse Primer (10nM) â 1ÂµL

10x Buffer â 5ÂµL

Q Solution â 10ÂµL

dNTPs (25mM) â 2ÂµL

Taq Polymerase â 0.5ÂµL

Water â to 50ÂµL

You have 2 samples plus a negative control that you need to test. How will you set up a master mix to make your pipetting more accurate and setting up this experiment go much faster than pipetting each individual component into 3 separate tubes? Remember to give yourself room for error.

While your PCR is running, you decide to go ahead and make your agarose gel. Your product size is expected to be 536bp. What percentage agarose gel do you need? How much agarose, and how much 1X TBE buffer to you need to accomplish this?

You pour your gel successfully, but use the last of the 1X TBE. You need more TBE to run the gel, but all we have is 50X TBE. You decide to make 3L of 1X TBE from the 50X TBE so that there is plenty for others (remembering your frustration from tissue culture and no PBS earlier in the day). How much 50X TBE do you need, and how much water do you need to make 3L?

Making Solutions:

You need 100mLs of a 5M solution of CaCl₂. The MW of CaCl₂ is 111 grams/mol. How many grams of CaCl₂ do you need?

You want 25mLs of the following solution: 0.5M CaCl₂ and 1M MgSO₄. You have 5M CaCl₂ and 2.5M MgSO₄. How much of each do you add to make your final solution?

Many times, you will be using someone elseâs lab notebook to formulate your own protocols. You will notice in some lab notebooks that the author will refer to nearly everything in volume rather than concentration. For example, it may say, âadd 5Î¼L DNA to the PCR tubeâ instead of âadd 500ng DNA to the PCR tube.â Which is more accurate? Why?

greymule951

2. After your frustration with tissue culture, you finally get your cells passaged and decide to set up your cDNA synthesis reaction, PCR, and agarose gel. You have extracted RNA from your cells, and now you need to proceed with the cDNA synthesis.

a. The first step is to determine the concentration of your RNA. You dilute your RNA 1:250, vortex it, move it to the cuvette, and run it on the spectrophotometer. The spec tells you that your concentration of RNA is 22Âµg/mL with an A260 of 0.32 and an A280 of 0.2. What is your ACTUAL RNA concentration, and what is its purity?

b. After determining your actual concentration of RNA, you are ready to make your cDNA. The protocol calls for the following for a total of 20ÂµL:

5x iScript Reaction Mix â 4ÂµL

iScript reverse transcriptase â 1ÂµL

Nuclease free water â x ÂµL

RNA template (100fg to 1ug total RNA) â x ÂµL

You decide to use 500ng (or 0.5Âµg) of RNA in this reaction. How many ÂµL of your RNA do you need, and how much water will you put in the tube?

c. You make the cDNA and get ready to set up your PCR for CD-10. The Qiagen protocol is as follows to a total of 50ÂµL:

cDNA â 2ÂµL

MgCl (25mM) â 4ÂµL

CD-10 Forward Primer (10nM) â 1ÂµL

CD-10 Reverse Primer (10nM) â 1ÂµL

10x Buffer â 5ÂµL

Q Solution â 10ÂµL

dNTPs (25mM) â 2ÂµL

Taq Polymerase â 0.5ÂµL

Water â to 50ÂµL

d. While your PCR is running, you decide to go ahead and make your agarose gel. Your product size is expected to be 536bp. What percentage agarose gel do you need? How much agarose, and how much 1X TBE buffer to you need to accomplish this?

e. You pour your gel successfully, but use the last of the 1X TBE. You need more TBE to run the gel, but all we have is 50X TBE. You decide to make 3L of 1X TBE from the 50X TBE so that there is plenty for others (remembering your frustration from tissue culture and no PBS earlier in the day). How much 50X TBE do you need, and how much water do you need to make 3L?

3. Making Solutions:

a. You need 100mLs of a 5M solution of CaCl₂. The MW of CaCl₂ is 111 grams/mol. How many grams of CaCl₂ do you need?

b. You want 25mLs of the following solution: 0.5M CaCl₂ and 1M MgSO₄. You have 5M CaCl₂ and 2.5M MgSO₄. How much of each do you add to make your final solution?

4. Many times, you will be using someone elseâs lab notebook to formulate your own protocols. You will notice in some lab notebooks that the author will refer to nearly everything in volume rather than concentration. For example, it may say, âadd 5Î¼L DNA to the PCR tubeâ instead of âadd 500ng DNA to the PCR tube.â Which is more accurate? Why?