BSM241 Lab 3
Isolation and analysis of chromosomal DNA from bacteria: Part 1
Introduction: In this series of exercises students will learn how to grow bacteria for the isolation of DNA, to isolate DNA using a commercial DNA purification kit (Promega) and to analyze the DNA product. To this end each student will learn how to use Nanodrop to quantify the amount of DNA isolated and to use agarose gel electrophoresis technology to visualize the isolated DNA.
Isolation of colonies and propagation of bacteria in liquid culture: The object of the first exercise is to learn how to aseptically inoculate one colony of bacteria into liquid culture to obtain a pure culture of genetically identical bacteria. Genetically identical bacteria that have been derived from one individual bacterium are known asclones. In order to ensure that all bacteria are genetically identical and free of other organisms each student must use aseptic technique. To this end students must wash down all working areas all pipettors, pull back their hair, roll up their sleeves and wash their hands and forearms. In some cases students are required to wear protective gloves. Students must not cough, sneeze, talk or move excessively over or around open test tubes, microcentrifuge tubes or Petri dishes. All tubes and Petri dishes must be open only for a given exercise; the students must work efficiently and quickly and should avoid leaving containers open for more than 1 minute. Caps and tops to Petri dishes must never touch the top of the lab bench. The instructor will demonstrate proper aseptic technique when handling bacteria.
Inoculation of bacteria into liquid media: The instructor has provided each student with a plate onto which Escherichia coli has been streaked for isolation to obtain individual colonies. Each student has a different strain and will be working with that strain and the DNA isolated from that strain for the duration of the semester. Each student will sample one colony and aseptically inoculate that colony into liquid media (Tryptic Soy Broth containing 30ug/mL of kanamycin and 10 ug/mL of tetracycline). The liquid media contains these two antibiotics to select for the growth of the particular E. coli strain with which each student is working and to ensure that only that strain is able to grow in the liquid media. The students will place the inoculated tubes into a 37oC shaking incubator to allow for the growth (multiplication) of bacteria to generate a dense culture where all of the progeny are identical to the parental bacteria. The instructor will allow the bacteria to grow for 24 hours and then store the cultures at 4oC until the next lab period.
Streaking bacteria for isolation: It is important to streak bacteria for isolation to ensure that one is able to sample one individual colony that represents a clone of the original single parental bacterium. Each student will learn how to streak bacteria for isolation from the plate that the instructor has provided to another plate. The plate onto which each student will streak their bacteria is Congo Red agar containing 30ug/mL of kanamycin and 10 ug/mL of tetracycline. The students will place the inoculated plates into a 37oC incubator. The instructor will allow the bacteria to grow for 24 hours and then store the cultures at 4oC until the next lab period. To review streaking for isolation please download and print off a protocol at the following website:
Terms to look up in your text book:clone, turbid culture, pure culture, aseptic technique, generation time and binary fission.
Isolation and analysis of chromosomal DNA from bacteria: Part 2
Isolation of total chromosomal DNA: Students will isolate chromosomal DNA from 1 mL of the overnight culture from the previous laboratory period. To this end students will use the Wizard (Registered) Genomic DNA Purification Kit. Please download and print off pages 1-3 and 16-18 from the following website for the protocol. The instructor has already harvested 1 mL of the overnight culture and stored the harvested culture at -80oC for this period. To this end the instructorpipetted 1 mL of the overnight culture into a 1.5 mL micro-centrifuge tube. A pellet of the cells was obtained by centrifuging the tubes containing the culture at high speed (~20,000 rpm) for 2 minutes. The supernatant containing the media was removed leaving only the cell pellet from which each student will isolate their DNA. Each student will get a chance to learn how to harvest bacteria for the isolation of DNA.
Harvesting bacteria: Observe the turbidity of the culture compared to the un-inoculated media. If each student only picked one colony this culture should represent multiple copies of bacteria identical to the parental bacteria. To learn how to harvest bacteria to obtain DNA each student will do the following during the 40 minute incubation time mentioned in step 5 below:
1. Add 1 ml (1000uL) of the overnight culture to a 1.5 mL microcentrifuge tube.
2. Centrifuge the culture at 20,000 rpm for 2 minutes to pellet the bacteria.
3. Carefully remove the supernatant containing the media in which the cells were grown without disturbing the pellet. The instructor will demonstrate this process.
4. Observe the size and appearance of the pellet.
Isolation of DNA using the Wizard Purification kit by Promega: Each student will follow the protocol that they have downloaded from the website to isolate our DNA. An abbreviated version of the protocol is mentioned below.
1. Obtain the micro-centrifuge tube containing a pellet of your overnight culture.
2. Add 600uL of the Lysis solution provided by the instructor. Use your P1000 pipette to gently pipette the pellet and solution up and down until you have a homogeneous mixture of harvested bacteria and lysis solution
3. Incubate the mixture at 80oC for 5 minutes and allow the mixture to cool down to room temperature on your desk top (~10 minutes are required to completely cool the contents of the test tube.
4. Add 3uL of RNase enzyme then gently invert the tube 5 times to mix the enzyme with the lysed bacteria (cell lysate)
5. Incubate this mixture at 37oC for 40 minutes and allow the mixture to cool down to room temperature on your desk top for 10 minutes.
6. Add 200uL of Protein precipitation solution (PPS) to the above mixture and then vortex vigorously at high speed for 20 seconds to mix the PPS solution with the cell lysate.
7. Incubate the sample on ice for 5 minutes.
8. Centrifuge the mixture at 20,000 rpm for 3 minutes.
9. Transfer 750 uL of the supernatant that contains the DNA to a 1.5 mL micro-centrifuge tube that already contains 600uL of room temperature iso-propanol. The pellet contains the cell membrane, cell wall and denatured proteins. The tube containing the pellet should be thrown out. Each student must be careful to avoid contaminating their supernatant with the pellet.
10. Gently mix the tube containing the DNA and iso-propanol by inverting the tube until white, thread like strands of DNA appear in the test tube. (invert the tube ~10 to 20 times)
11. Centrifuge the mixture at 20,000 rpm for 2 minutes.
12. Carefully pour off the supernatant and drain tube upside down on absorbent paper provided by the instructor.
13. Add 600uL of room temperature 70% Ethanol to the DNA pellet and gently invert 10 to 20 times to clean the DNA pellet.
14. Centrifuge the test tube at 20,000 rpm for 2 minutes to ensure that the DNA pellet is again at the bottom of the micro-centrifuge tube.
15. Carefully pour off the ethanol and remove the rest of the ethanol by using a P200 pipettor. Make certain not to disturb the pellet. The instructor will show you how to do this.
16. Allow the tube to air dry on absorbent paper for 10-15 minutes.
17. Add 100uL of DNA rehydration solution (RHS) to the pellet and allow the DNA to dissolve in the solution by incubating the solution at 4oC until the next period.
Study questions: Use your text book, online sources, the Promega instruction manual and the pre-lab lectures to answer the following questions.
1. What does the lysis solution contain? In your own words describe how this lyses the bacteria.
2. Why isn’t lysozyme required to lyse the Gram negative bacteria that we are using in this lab while it is required to lyse Gram positive bacteria.
3. What is the purpose of adding RNase?
4. What does the Protein precipitation solution contain? In your own words describe why this solution is able to precipitate proteins and cell debris but not the DNA.
Quantifying total chromosomal DNA using Nanodrop spectrophotometry: Once one has isolated DNA it is important to determine how much DNA has been isolated in order to use that DNA in subsequent applications. For example, one must know the amounts of DNA present in ng/uL or ug/mL for applications like restriction digests and cloning or pcr (polymerase chain reaction).
Visualizing total chromosomal DNA using Agarose gel electrophoresis: Once one has isolated DNA it is also important to examine the quality of that DNA. DNA should show up on an agarose gel primarily as distinct bands not as a large smear. If the DNA shows up as a long smear on an agarose gel that means that the integrity of the DNA has been compromised during the purification process.
1