2. After your frustration with tissue culture, you finally getyour cells passaged and decide to set up your cDNA synthesisreaction, PCR, and agarose gel. You have extracted RNA from yourcells, 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, andrun it on the spectrophotometer.   The spec tells youthat your concentration of RNA is 22µg/mL with an A260 of 0.32 andan A280 of 0.2. What is your ACTUAL RNA concentration, and what isits purity?

b. After determining your actual concentration of RNA, you areready to make your cDNA. The protocol calls for the followingfor 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) ofRNA in this reaction. How many µL of your RNA do you need, and howmuch 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 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 negativecontrol that you need to test. How will you set up a master mix tomake your pipetting more accurate and setting up this experiment gomuch faster than pipetting each individual component into 3separate tubes? Remember to give yourself room for error.

d. While your PCR is running, you decide to go ahead and makeyour agarose gel. Your product size is expected to be 536bp. Whatpercentage agarose gel do you need? How much agarose, and how much1X TBE buffer to you need to accomplish this?

e. You pour your gel successfully, but use the last of the 1XTBE. 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 isplenty for others (remembering your frustration from tissue cultureand no PBS earlier in the day). How much 50X TBE do you need, andhow much water do you need to make 3L?

3. Making Solutions:

a. You need 100mLs of a 5M solution of CaCl₂. The MWof CaCl₂ is 111 grams/mol. How many grams ofCaCl₂ do you need?

b. You want 25mLs of the following solution: 0.5MCaCl₂ and 1M MgSO₄. You have 5MCaCl₂ and 2.5M MgSO₄. How much of each do youadd to make your final solution?

4. Many times, you will be using someone else’s lab notebook toformulate your own protocols. You will notice in some lab notebooksthat the author will refer to nearly everything in volume ratherthan concentration. For example, it may say, “add 5μL DNA to thePCR tube” instead of “add 500ng DNA to the PCR tube.” Which is moreaccurate? Why?