LAB 1: AN INTRODUCTION TO MICROVOLUMETRICS AND PIPETTING

Transcription

1 Name: Book # Per. Name: Name: Book # Book # LAB 1: AN INTRODUCTION TO MICROVOLUMETRICS AND PIPETTING PRELAB: 1. Approximately 28 drops of liquid, from a medicine dropper or disposable pipette, equals 1 ml. Each drop then represents what part of a milliliter (ml)? Show work. 2. Approximately how many ul are found in one drop of liquid. Remember 1000 ul = 1 ml 3. Four hairs, found at a crime scene, can yield about 1 ug of DNA. How many ug of DNA can be extracted from just one hair? 4. How many nanograms does this represent? 1000 ng = 1 ug 1

2 CONCLUSIONS FOR LAB 1: Draw the gel upon completion (use colors) 1. The dyes that you separated using gel electrophoresis were: Orange G (yellow color, molecular weight = ) Bromophenol blue (purple color, molecular weight = ) Xylene cyanole (blue color, molecular weight = ). What electrical charge did these dyes have and what evidence made you come to that conclusion? 2. Molecular size can play a role in separation with small molecules moving through the gel matrix more rapidly than larger molecules, but other properties can effect their movement as well. From your results, did it appear that these molecules were separated clearly on the basis of size? If not, explain what other factors may have played a role? 3. Which tube contained a single dye? What was the name of the dye? 4. Why is it important to actually see the solution enter the pipette tip? 5. Which stop do you use when dispensing (expelling) a solution from the micropipette? 6. Which stop do you use when preparing to aspirate (draw up) a solution into the micropipette? 7. After loading your gel, did any solution remain in tubes A, B or C? If so, what could account for that? 2

3 LAB 2: RESTRICTION ANALYSIS OF para AND pkan-r PRELAB: 1. Diagram the two plasmids below. Be sure to identify: names, restriction sites, total # of base pairs of each plasmid and any gene locations. 2. What are the functions of the following genes: a. amp r b. kan r c. ara C d. rfp 3. Which antibiotic resistance giving gene is found on: a. para b. pkan-r 4. One of the plasmids has been engineered to ensure the expression a neighboring gene s protein. Which of the plasmids has been engineered for protein expression? 5. What organism did the DNA for the mutant fluorescent protein originate? 6. Summarize, in a brief list, what you will be doing in this lab today: 7. Why do you have two microfuge tubes without any restriction enzymes? 3

4 CONCLUSIONS FOR LAB 2: BamHI and HindIII are specific restriction enzymes that will cut the DNA wherever it encounters the six-base recognition sequences indicated below. Remember that DNA is anti-parallel, one strand runs from the 5 to 3 direction and the other complementary strand runs from the 3 to 5 direction. Careful examination of the restriction sequences will reveal that the sequence of nucleotides in recognition sequences is always found to be a palindrome; that is to say, it reads the same on both strands when read in a 5 to 3 direction (ex. race car). When the restriction enzymes encounter the specific recognition sequence, it will cut the DNA helix leaving four unpaired bases forming a sticky end. As demonstrated, the sticky end for HindIII in the 5 to 3 direction is: AGCT. When complementary sticky ends are encountered they can be spliced together! 1. In a 5 to 3 direction, what sequence of bases represents the sticky end for BamHI? 2. Examine the para and pkan-r plasmid maps and complete the following: a. para digestion will yield total fragment(s) with bp length(s) of: b. pkan-r digestion will yield total fragment(s) with bp length(s) of: 3. Will we, based on the types of restriction enzymes used, be able to mix and match fragments of both plasmids together? Why or why not? 4

5 LAB 3: LIGATION OF para/pkan-r RESTRICTION FRAGMENTS PRODUCING A RECOMBINANT PLASMID, para-r PRELAB: 1. Briefly define the term recombinant DNA? 2. What does the term ligation indicate? 3. In order for two sticky to join together, what relationship needs to exist between them? 4. Looking at the directions (Methods) for Lab 3, what is the first step and why is it so important? 5. Why are you using solutions from the A+/K+ microfuge tubes instead of the A-/K- tubes? 5

6 CONCLUSIONS FOR LAB 3: 1. What would have happened if we omitted step one of the lab? 2. Although many possible recombinant plasmids could have been formed, draw and label three possibilities (combinations of the four different fragments created). Make sure one of the possibilities is the plasmid we are trying to form: 3. In the DNA molecule, there are two types of chemical bonds, covalent and hydrogen bones. Answer the following questions relating to these bonds: a. Which bond is the strongest? weakest? b. Which bond forms first? Between what molecules? c. Which bond forms second? Between what molecules? d. Which bond requires DNA ligase? 6

7 LAB 4: Confirmation of Restriction and Ligation Using Agarose-Gel Electrophoresis PRELAB: 1. Analyzing the gel to the right, list the order of the DNA fragments from largest to smallest: 2. Why is it important that we confirm that we have digested para and pkan? 3. Describe the three forms/types of uncut plasmids that may be found in our gels: supercoiled, open-circle and multimer: 4. Besides using gel electrophoresis to separate DNA fragments according to their sizes, it can also be used to ESTIMATE THE ACTUAL SIZE, in base pairs, of each fragment. To do this we need to have a control/marker, a solution that contains known fragment lengths loaded into our gel as well. We can then compare our DNA banding patterns to the positions of the known fragment lengths to estimate the size of our fragments. We can then confirm if our plasmids were cut and additionally if they were cut in the right places. The marker we will use has 10 known sizes of DNA fragments, which I have positioned on the gel below. Using what you know about the plasmids, position gel bands in their predicted locations. I have done the first one for you: 7

8 CONCLUSIONS FOR LAB 4: 1. Attach a picture/print-out of your gel at the bottom of this page. 2. For para: a. Does your gel reflect that you cut your plasmids in the A+ tube? b. What evidence reflects your conclusion for 2a? c. Did you cut para in the correct location? d. What evidence reflects your conclusion for 2c? 3. For pkan-r: a. Does your gel reflect that you cut your plasmids in the K+ tube? b. What evidence reflects your conclusion for 3a? c. Did you cut para in the correct location? d. What evidence reflects your conclusion for 3c? 4. Does it appear that you formed newly ligated plasmids? Please describe the evidence that supports your conclusion. 5. Do you see evidence of the three plasmids types/forms (multimer, open-circle and supercoiled) in your uncut (K-/A-) lanes? 6. Two of the 702 bp pkan-r fragments, rfp gene, may form a circularized plasmid because each end of the fragments terminates in BamH I and Hind III sticky ends. Is there evidence of a circularized 1404 bp plasmid in the ligated lane? [Gel photo goes here, tape neatly] 8

9 LAB 5: Transforming Escherichia coli with a Recombinant Plasmid PRELAB: 1. Briefly describe the purpose(s) of each step required for constructing your recombinant plasmid from para and pkan-r restriction fragments: a. Lab 2: b. Lab 3: c. Lab 4: 2. What is transformation in our lab? 3. What does it mean if cells are competent? 4. Based on the procedures of Lab 5, how would you know if a bacterium was transformed with a plasmid that contained the amp r gene? a. What is the size of the fragment that carries the amp r gene? b. From what plasmid did the amp r gene originate? 5. How is the P+ culture of bacteria treated differently than the P- culture? 6. Why did we bother with the P- culture at all? 9

10 CONCLUSIONS FOR LAB 5: 1. Predict the growth, if any, on the following plates. Put a + on the plate(s) if you expect growth and (-) if you do not expect growth: 2. What do ALL the cells growing on the LB/amp and LB/amp/ara plates have in common? 3. What gene must be found in plasmids these bacteria contain? 4. What data provides evidence that the bacteria used in this lab did not just naturally have that resistance? 5. Would you expect that all the cells growing on the LB/amp/ara plate were transformed with the exact same plasmid? Why of why not? 6. Could any of the bacteria growing on the P+ portion of the LB plate: a. Contain amp r? Why or why not? b. Contain the rfp gene? Why or why not? c. What further experimentation could you do to substantiate your answers to 6 a/b? 10

11 FINAL RESULTS: Transformed Escherichia coli with a Recombinant Plasmid 1. Complete the table below (+/-) to compare how your actual transformation results differed from your predicted results. Plate LB LB/Amp LB/Amp/Ara Predicted Results Actual Results P- P+ P- P+ 2. Did your results differ from the predicted results? If so, how? 3. Did you get any red colonies on your LB/Amp/Ara plates? If so, how many? 4. Why did red colonies only appear on LB/Amp/Ara plates? 5. Concerning the bacteria found on the LB/Amp plate: a. Could some have been transformed by para-r? b. If so, what experiment could you perform to support your hypothesis? 6. Which part(s) of this lab did you find to be the most interesting to you? (Answer individually for this question.) 11