Anthony Araracap Period 4 AP Biology (Lab 7A and 7B Lab Report) Restriction Digestion and Analysis of DNA (7A) and DNA

Fingerprinting (7B) Background: Gel electrophoresis is a technique used to analyze fragments of DNA. The restriction enzymes cut the DNA into numerous fragments. Once fragments are generated, they separate from each other using gel electrophoresis. E.Coli is bacteria used for light. Bioluminescence is when an organism creates own light through a chemical reaction, while fluoresce is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. Ampicillin is an antibiotic used to kill bacteria. Purpose: The purpose of the lab was to cut DNA using restriction enzymes, separate DNA fragments using electrophoretic means. Also, to determine the number of base pairs in fragments of DNA. Hypothesis: (Part A) If prepared digested DNA samples undergo gel electrophoresis, then the number of base pairs in fragments of DNA can be determined. (Part B) If we insert a plasmid, then the transformation of colonies will be successful. Materials: ● Restriction Digestion and Analysis of Lambda DNA kit ● ice bath ● 37 degrees C water bath ● 65 degrees C water bath ● micropipettes ● electrophoresis chamber ● Agar plate containing LB/ No Amp ● Agar plate containing LB/AMP plates (red mark on side) ● Sterile inoculating loop ● Plastic transfer pipettes ● Clear 2.0 mL micro tubes ● Cotton swabs ● Sharpie ● Piece of tape for sealing plates ● waste container ● styrofoam cup with ice ● blue 1.5 mL tube of CaCl2 ● clear 2.0 mL tube of plasmid DNA labeled either PM1 or PM2 ● hot water bath at 42 degrees C ● starter bacterial plate Procedure: ● (Followed Quick Guide-Restriction Digestion and Analysis procedures & Bacterial Transformation procedures)

Data & Observations: During this lab, several observations were made. First off, each colored microtube was a different color. Yellow tubes were labeled L for lambda DNA, Violet tubes were labeled P for Pstl digest, Green tubes were labeled E for EcoRI digest, and Orange tubes were labeled H for HindIII digest. TAE was also used in the lab as a buffer. Another observation was that the bacteria turned blue due to the fluorescent protein. Also, the plates had each transformed the bacteria, as multiple colonies were grown, although not all may have been grown. Also, not all the gels produced were readable. Besides this, the starter plate had a weird odor for the second part of the lab. Furthermore, when the plates that had produced bacteria were placed under UV light; each group yielded various colors, from green to pink.
Marker Lambda DNA (no enzyme) Base Pairs 23130 9416 6557 4361 2322 2027 Distance (mm) 12 Base Pairs 37000 Lambda with PstI Lambda with EcoRI Base Pairs 7000 Distance (mm) 18 Base Pairs 9000 Lambda with HindIII Distance (mm) 16 Base Pairs 9000

Distance (mm) Band 1 Band 2 Band 3 Band 4 Band 5 Band 6 15 16 19 24 36 39

Distance (mm) 20

Analysis: Error Analysis: Throughout this lab several errors may have occurred. For instance, using the incorrect amount of agarose (0.5g). Another error may have been not mixing the components in the tube in order to get the liquid at the bottom of the tube, yielding less accurate data. Also, when inserting the DNA into the gel slots, some may not have been carefully placed in, causing inaccuracy in the ladder.The person transferring had to have a steady hand and good eyes so that the gel wasn’t poked and the DNA made it into the chamber without problems. Another error may have been that the wrong DNA samples were added to the wells, but the right ones were identified and later labeled correctly, out of order. The amounts of the different solutions could have been distorted. Also, there were many lumps in the agar poured into the plates. The spreading rod that was used to spread the bacteria onto the agar could have been too hot when it was used to spread, and killed the bacteria, or while the lid of the petri dish was off, some other contamination from the room could have infected the bacteria causing different results. Finally, another place of error could have been in setting of the bacteria on the plates. Discussion: The hypothesis for part A of this lab was “If prepared digested DNA samples undergo gel electrophoresis, then the number of base pairs in fragments of DNA can be determined” was accepted. Using the base pairs and distance for the DNA L, P, E, and H were able to be graphed once the lab was

concluded. Once the samples were prepared, they underwent agarose gel electrophoresis in the refrigerator. After, the movement of the restriction enzymes were observed. The genetic marker in the lab yielded both large and small fragments (base pairs of 23,130 and 15 mm traveled to 2027 base pairs and 39 mm traveled). Furthermore, the L observed in the gel only had one fragment which was 35,000 base pairs, the P had another fragment; 7000 base pairs, the E and H also had 1 fragment that was 9000 basepairs. As the distance grew bigger, the base pairs were less. Then, after each procedure for part B was completed, bacteria was placed in UV light to be observed. Some of the petri dishes had shown that transformation of bacteria had occurred, thus allowing the plasmid to survive because it was recombinant.The petri dish with LB/Amp (+) was expected to have many transformed colonies and the results reigned true. Also, in the colonies, tiny dots of bacterial colonies that were green could be observed. In the second petri dish with LB/Amp (-) green fluorescent dots were seen, but significantly less than the first dish. In the last petri dish with LB/No amp (-) only a few tiny fluorescent green circles present.The hypothesis “If we insert a plasmid, then the transformation of colonies will be successful” for part B was then accepted because colonies were successfully transformed, as seen in the actual plate results, thus both hypotheses were accepted for part A and B. Discussion Questions: (Pre-Lab [A]) 1. a. Restriction enzymes cut DNA into numerous fragments, acting like scissors. b. DNA has a negative charge and will move in the positive direction in an electrical field. c. The movement of DNA fragments in the gel is effected by due to the negative charge of DNA. d. Larger fragments move much slower through the gel and thus on a DNA ladder the larger portions will be near the top. e. The genetic marker in this case acts are the control. 1. The largest fragment will be D. 2. The smallest fragment would be B. 3.


4. The large fragments would be near the top of the gel since it is more difficult for larger pieces to be strained through the gel. 5. There would still only be 4 bands present. 6. Fragment D would be heaviest because it is the largest piece of DNA and would have the largest mass. 7. Each enzyme produces different sizes of restriction fragments that have different patterns, which suggest different enzymes are cutting at random locations. (Post-Lab [B]) Plate # Plasmid Present? Amp present in plate? Actual Plate results(drawing) #1 LB/Amp (+) (red line) #2 LB/Amp(-)(red line) YES YES NO YES #3 LM/No Amp(-)(no red line) NO NO

Actual Plate results(description)

specks of bacteria, some colonies weren’t transformed

larger colonies with small colonies

not much visible, possibly tiny specks of bacteria

1. In plate 1 there were about 800 colonies, plate 2 had about 30, and plate 3 had around 20. 2. The plasmid mix we had was EGFP. The three colors of fluorescent bacteria that were observed were blue, dark green, and light green. 1. Colonies were able to be different colors due to the green fluorescent protein, which is used for its bioluminescence; produced when the gene is expressed and energy is transferred to it within its host. 2. The plasmid was able to enter the cell through the bacteria and plasmid DNA solution with calcium chloride, which neutralized the negative charges. 3. If a group did not get any transformed colonies it may have been due to the mixture not being placed in the ice bath quickly enough, or incorrect mixtures of the DNA once the restriction enzymes were added.

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