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 22µg/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 20µL:
5x iScript Reaction Mix – 4µL
iScript reverse transcriptase – 1µL
Nuclease free water – x µL
RNA template (100fg to 1ug total RNA) – x µL
You decide to use 500ng (or 0.5µg) of RNA in this reaction. How many µL 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 50µL:
cDNA – 2µL
MgCl (25mM) – 4µL
CD-10 Forward Primer (10nM) – 1µL
CD-10 Reverse Primer (10nM) – 1µL
10x Buffer – 5µL
Q Solution – 10µL
dNTPs (25mM) – 2µL
Taq Polymerase – 0.5µL
Water – to 50µL
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?
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 else’s 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, “add 5μL DNA to the PCR tube” instead of “add 500ng DNA to the PCR tube.” Which is more accurate? Why?
Sample dilution = 1:250
Concentration of the diluted sample = 22 ug/mL
Concentration of the original sample = 250 X 22 = 5500 ug/mL = 5.5 ug/uL (This much RNA concentration is unusual and rather rare)
A260/280 = 0.32/0.2 = 1.6
A260/280 ratio close to 1.8 is considered as pure.
So, the given sample has some contaminants such as trizol.
Required RNA concentration = 0.5 ug
1 uL ----> 5.5 ug
? uL -----? 0.5 ug
= 0.09 uL
Total reaction volume = 20
All the components = 4 + 1 + 0.09 = 5.09
Volume of water = 20 - 5.09 = 14.91 mL
We have to set for 2 samples and a negative control. So, to make
up for error, we should calculate for 3.5 samples.
cDNA = 2 uL
MgCl2 = 4 uL X 3.5 = 14 uL
FP = 1 uL X 3.5 = 3.5 uL
RP = 1 uL X 3.5 = 3.5 uL
Buffer = 5 uL X 3.5 = 17.5 uL
Q solution = 10 X 3.5 = 35 uL
dNTPs = 2 X 3.5 = 7 uL
Taq = 0.5 X 3.5 = 1.75 uL
Water = 24.5 X 3.5 = 89.25 uL
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