Today s Lecture. Review of events for this week Background material so you can understand DNA extrac?on and PCR Quiz

Transcription

1 Today s Lecture Review of events for this week Background material so you can understand DNA extrac?on and PCR Quiz

2 This week Con?nue purifying colonies and start a liquid overnight culture Extract DNA Several things due

3 Due this Weds: Water Quality Exercise 1. There are two specific tables to prepare: 1. MPN Analysis results 2. Membrane filtra?on analysis results 2. Paragraph discussing why M tec agar allows enumera?on of E. coli Again, please hand in something that is well wriren, clear, no spelling or grammar mistakes Numbering is off in assignment, accdentally numbered as 4 3. Extra credit, do 2 above for mfc agar

4 Title Due next Monday: Food Lab Report Provide a specific title that is descriptive. You will want to include the food you analyzed as well as what you compared it to. Introduction (~1-2 pages) Describe what food contamination is, and some current issues. You must include at least one specific example related to microbial contamination of food (does not have to be your food!). This could be a published paper that describes a specific type of bacteria known to be found in food; epidemiological data about food borne illnesses; toxins associated with food-borne pathogens; etc. You must include your source of information and it has to be a published scientific paper (i.e. journal article of some kind)

5 Food lab report II Methods (~1-2 pages). Describe what samples were collected; how they were prepared; the media used; the methods calculating CFU/gm food; and how data was analyzed. Results (~1-2 page written plus ~1 page Figures/Tables): The goal of this section is to present your findings and also those of the entire class (both rooms). You will need Tables and Figures to accomplish this. A table is a good way to show all the data and the actual numbers, while a figure such as a bar graph is a good visual way for your reader to compare the data. Please present your data in two ways that make the most sense to you. Each Table or Figure should have a legend that describes what data is shown in each. This means you will have two Figures or Tables. In the text, describe the class findings, and refer to each table or figure.

6 Food lab report III Discussion (~1-2 pages) Follow the general laboratory guidelines. In addition, address these points in your discussion: 1. Start out with a description of what you found--how did the density of bacteria and variety differ between food types, sources and treatments. 2. Describe what types of bacteria are known to associate with one of the food items, by searching Pubmed, the Foodnet (CDC) or FoodHACCP websites. If your specific found cannot be found, use the closest relative. Provide at least two examples of bacteria associated with your food, and either pathogen or non-pathogen is fine. Cite your information sources. For this, you may use a website as your source, but it has to be a good quality one. 3. Describe a follow up experiment. References Site your sources according to the general guidelines.

7 Food Lab report IV From the general guidelines: References: This section is simple. You list your sources of information used throughout your lab report. There are several formats to follow. However, you will receive an example journal article. Please follow the format of that paper. The only acceptable citations are scientific works published in journals or books. No http or URL citations. You can use things like wikipedia to give you background, but you must use a text book or journal article for the information you present, unless you are granted an exception by Professor Ottemann. Figures/Tables: Attach each figure or table on a separate sheet of paper at the end of the report with one page per figure or table. Each figure/table should be numbered consecutively and must have a caption and description of any ambiguous information--e.g. abbreviations

8 A note about plagiarism Keep notes of the resources you ve used to get your ideas Do not direct quote, instead you must paraphrase and then put a cita?on example on next slide. A cita?on is a descrip?on of a book, ar?cle, URL, etc. that provides enough informa?on so that others can locate the source you used themselves. Failing to properly cite your sources gives the appearance that you are claiming the work of others is actually your own is called plagiarism and is a serious viola?on of University rules. Good resource for how to conduct literature research: hrp://nerrail.ucsc.edu/index.html

9 Example of what we want to see: NOT: According to Smith et al., Listeria monocytogenes is a serious concern in so` cheeses but has not yet emerged as a pathogen associated with fruits (Smith et al., 2001). YES: Listeria monocytogenes can be found in foods such as so` cheeses, but was thought to generally not occur in foods such as fruits (Smith et al., 2001). This concept was challenged, however, in 2011 when a large outbreak occurred in cantaloupes (CDC a, 2011).

10 To help you with your wri?ng: Food Lab Report is due 10/31 This version will count toward 80% of your grade We will provide feedback by Weds 11/9 and you will get a chance to revise and hand in again (11/14/11) The revision will count toward the remaining 20% of the food lab report grade. You will be graded on the quality of your response to our sugges?ons

11 This week we start Molecular Strain Iden?fica?on Goal is to use 16S rrna gene analysis to determine the iden?ty of your unknown strain. How is this done? Isolate pure single colonies (done last week and today) Start overnight liquid culture on Tuesday Select three colonies/group of two for liquid culture Extract chromosomal DNA (Weds) Analyze chromosomal DNA and set up PCR for 16S rrna gene (Day 2, Next monday) Analyze PCR products next Weds (11/9) Restric?on Fragment Length Polymorphism and Sequencing Sequencing and bioinforma?cs analysis (11/21)

12 Weds: Ex 10 part 1 (Today Monday we will restreak single colonies again) Tomorrow you need to go into lab either 7:30AM 1 PM or 4 5 PM 1) Each person needs to start two overnights 1) Find two single colonies that each came from a different original bacterium mark them on the back of the plate. 2) 3 tubes that each have 3 ml of LB. Label each tube with your ini?als and something so you will know which colony is which 3) Gloves, lab coat and eye protec?on as this is BSL2 work 2) Using a sterile s?ck or cooled loop, touch the first colony 3) Place it into tube 1, using sterile technique 4) Also do a no bacteria control, where you put either the loop or s?ck into the LB, without touching the bacteria this will ensure that all is sterile 5) Place in shaking 37ºC incubator 6) Clean your bench and wash your hands

13 Weds: Extract Chromosomal DNA 1. Mul? step so you have to keep careful track 2. Lyse the cells. 3. Purify away the membranes, proteins, and polysaccharides. 4. Remove the RNA. 5. Analyze the DNA on 10/31: (1) visually DNA on an agarose gel by (2) measure concentra?on by UV spectrophotometry.

14 Extract Chromosomal DNA 1. Each person should do one extrac?on, so pick on of your two colonies 2. Mix 350 µl of the rest of the culture with 150 µl glycerol in an eppendorf tube to make a frozen stock. Label with room number, drawer number and year. 3. Take 1.2 ml and collect bacterial cells by centrifuga?on make sure to balance tubes! 4. Add lysis enzymes to lyse the cell wall: lysozyme and then SDS Incubate as suggested 5. Remove RNA with an enzyme called RNAse 6. Precipitate the protein (ammonium acetate) and remove this + cell wall by centrifuga?on 7. DNA is le` over in supernatant 8. Precipitate DNA using isopropanol followed by wash with 70% ethanol.

15 Next period: Check to be sure you got Genomic DNA Run on gel Some modifica?ons to lab manual 50 ml agarose instead of 75 ml Using Gel Red instead of Ethidium Bromide (2.5 ul) Image from:

16 Quan?fy your genomic DNA 1 UV Spectrophotomer DNA/RNA absorbs strongest at 260 nm 50 µg/ml has OD of 1 at 260 nm. You can use this rela?onship to calculate DNA concentra?ons of an unknown samples. Pure DNA has a 260/280 of 1.8 Abs wavelength

17 A`er you know you have genomic DNA, the next step is to set up your PCR 10/31 we run the gel, check the genome and set up the PCR