Practical Exam -Be able to do these • Scale & Pipette
• Serial Dilutions & Standard Curve (1:2 dilution)
• -Identify different laboratory tools
• -Spectrophotometry of DNA
• -Loading a gel
• -Affinity Chorography Scale & Pipette How to tare a scale
How to read the scale correctly
How to read all three pipettes
Note: your lab manual is a great review source
Serial Dilutions & Standard Curve Review how to do a (1:2) serial dilution
*** remember to throw out the last 100 microliters
from the 2nd to last well
Every well should have a total of 100 microliters at
the end of your dilution
The blank should be your last well
Identify laboratory tools Pipette (which range?) • Roller pipette • Centrifuge Spectrophotometer • Microcentrifuge tubes Blue/yellow tips • Heat block • Gloves Test tube rack • Glass tubes • Cuvettes Scale Spectrophotometry of DNA Know the different wavelengths, especially which wavelengths DNA and Protein absorb the most
Remember that absorbance is directly related to
concentration
Know how to blank the machine
Gel Loading Skillz Step one: Breath. It’s not that bad. Just go slow. Steady your hand with the other hand on your wrist, if necessary Go all the way into the well before pumping out the liquid— don’t lose all your sample because it floated away over the top of your well. Inject sample SLOWLY—go too fast and it will mushroom out DON’T stab through the gel. This will lose you points. Don’t go to the second stop—don’t want bubbles Make sure to not release the plunger until you pull it out of the gel, or you will draw your sample back up accidentally. Practice the motion, holding your thumb down until you are out of well. Affinity Chromatography Review the steps to affinity chromatography, pay attention to the specific vocabulary like the different buffers, what ‘elution’ means, and what the ‘flow through’ is. Written Exam -Take in the testing center anytime during the test week Reading a Gel Know how to read band size compared to a ladder
Understand where longest/shortest fragments
appear
Know the mechanics of DNA migration—why does it
move? DNA fingerprinting Understand what a restriction endonuclease is, and how we use them
Know the possible applications: crime scene
investigation, species identification, paternity determination, ancestry identification, creating evolutionary trees… In each case, the ability to compare individuals based on DNA sequence gives important information. For example, a crime suspect may have the same DNA fingerprint as a sample from the crime scene. ELISA What are we testing for? –antigens…which are? – control will not have antigen ( will be clear) + control will have antigen ( will be colored) 1st add primary antibody (binds to antigen of interest) : If it doesn’t bind then that means that there is no antigen to bind to. 2nd add secondary antibody (binds to primary antibody): HRP enzyme is already on the secondary antibody HRP enzyme: Converts the substrate into a visible color for detection (HRP enzyme + substrate = color) 3rd add the Enzyme substrate (converted by the HRP enzyme into a visible color for detection) ELISA (cont.) Affinity Chromatography What is the purpose? –Isolate/purify a protein Know what a ligand is. Just do it. NOTE: Ligand is bound to matrix—never comes out in any flow-through Understand what comes out in each flow-throw— protein of interest does NOT come out on first flow- through. That would utterly defeat the point and probably destroy the universe. Second step: wash the column (get stuck things off) Third step: elute the column (remove protein of interest) Understand: contamination, size of protein in band (where are the big/small bands?), How pure is the sample? Affinity Chromatography (cont.) (*Don’t worry about desalting) DNA Quantification SDS denatures proteins and biological membranes, and adds overall negative charge Protease K destroys proteins, allows DNA to be broken free from cell
EDTA chelates divalent metal cations (Mg2+) and
thus inhibits DNAses from destroying your DNA. Very important.
SDS and protease K can also help inhibit DNAses.
[DNA] and %DNA Wavelength Absorbance DNA was extracted from a 600 mg sample of A260 .70 fish and allowed to air dry. It was then A280 .2 resuspended in 400 µl TE buffer. A 1:5 A234 .34 dilution of the resuspended DNA- was made and then analyzed in the spectrophotometer. A320 .019 Discuss the purity of this sample using ALL of What is the concentration of DNA in the the wavelengths tested. resuspended sample? A260 * (known [DNA]) * Dil. Factor = [DNA,µg/ml] A260/A280 = 0.70/0.20 =3.5– Evidence for 0.7 * 50 μg/ml * 5 = 175 μg/ml clean DNA-no proteins (<1.7) A234/A260 = 0.34/0.70 =0.485– Evidence for What is the percent of DNA in Fish tissue? clean DNA-also no proteins (>.5) A320/A260 = 0.019/0.70 =.027– The cuvette [DNA, µg/ml] * (resuspension TE) = amount DNA, µg was clean and free from particulates (>.05) 175 μg/ml * 0.4ml = 70μg Amount DNA/amount tissue * 100 = %DNA *****DNA: there is a known standard for 70 μg _ * 100% = 0.0116% A260 (absorbance value). A reading of 600,000 μg 1.0A at A260 = the DNA is 50ug/ml DNA Quantification (cont.) REMEMBER TO ACCOUNT FOR ALL THE FLUID, BLAST IT! But really, we don’t give a rat’s fanny about anything except the DNA—so make sure you account for liquid used to suspend, and any dilutions. That’s all the math is doing, anyway. Review that. It might help you remember why you’re multiplying/dividing where and when you are. Protein Quantification We us the spectrometer to find the amount of protein present, just like DNA—but different wavelengths. The Dye (coomassie brilliant blue) attaches to the protein (at free NH3+), and the dye/protein complex gets shifted to a higher absorbance value (465 to 595). –this means we can read it without worrying about other things absorbing light at that higher wavelength (because hardly anything else in your solution will absorb that frequency). Protein Quant. Example A Protein Quantification experiment was Tube # (X) A 595 Reading (Y) performed similar to the one you did in the laboratory. 0 µg 0 20 µg BSA 0.080 0.3 g of a powder was dissolved in PBS to yield a total volume of 5 ml. Since the protein 40 µg BSA 0.150 was dissolved together with water to total five milliliters of volume, you will not need to 50 µg BSA 0.175 account for a volume change due to the Sample replicate 1 0.160 powder. Sample replicate 2 0.165 The dissolved protein sample was diluted 1:10. X*X X*Y Two duplicate samples were prepared for 0 0 quantification using 20 µl of diluted sample protein, 75 µl of PBS and 5 ml of Coomassie 400 1.6 Blue Dye. 1600 6 2500 8.75 Determine Slope: = .00363 Total= 4500 Total= 16.35 ∑(X*Y)/∑(X*X) total= Slope = .00363 .00363 Example (cont.) 1. Amount of protein as measured in the 2. Concentration of protein in the 1:10 diluted cuvette : sample: Y=mX (Use Average reading for Y --add both sample (divide the amount in the sample by the readings up and divide by 2) Y = 0.1625 volume of the sample) µg / volume OD = m * µg 44.76 µg / 20 µl = 2.238µg/µl 0.1625 = 0.00363 * x x = 44.76 µg 4. Determine the amount of protein present in the 0.3 g powder sample: 3. Concentration of protein in the original dissolved sample (in the 5 ml sample): Convert the units to be compatible with the total volume in ml: (Multiply the diluted sample concentration 22.38 µg/µl = 22.38mg/ml by the dilution factor) Then multiply by 5ml to cancel out the fluid 2.238µg/µl *10 = 22.38g/l and be left with amount of solid in mg: 22.38mg/ml * 5ml = 111.9 mg Info from your packets: What is SDS PAGE? Why the proteins Why is protein concentration by move? What determines how proteins spectrophotometry harder than move in the gel? determining DNA concentration? Which one requires a standard curve, and why? Proteins are first denatured by exposure to SDS. SDS denatures proteins by Since proteins are not made of a competing for hydrophobic bonds and uniform mixture of amino acids a also gives the protein a uniform negative constant relationship between protein charge. Adds a uniform negative charge. concentration and absorbance at a (without the – charge it wouldn’t do specific wavelength does not exist. Thus anything. It wouldn’t move) (DNA proteins standards must be used with doesn’t need SDS because the DNA some means of measuring the amount already has a – charge.) of protein like coomassie blue. The denatured protein is then placed in A constant relationship does exist for an electric field and migrates towards absorbance of DNA at 260nm, thus a the positive pole. Larger proteins take standard curve is not necessary if the longer to make their way through the DNA is of high enough purity. pores of the gel, while smaller protein make their way more easily.