MCB 251 UIUC Unknown Insert Mode Lab Report

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MCB 251 UIUC Unknown Insert Mode Lab Report

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IE #3 Lab report Analysis of Unknown Insert Mode #1 MCB 251 SP20 IE #3 Analysis of an Unknown Insert Mode of analysis #1 Lab Report (85 points) Due April 24th by 11:59 pm The lab notebook report for mode #1 analysis of your unknown insert should include: • The purpose of the experiment, all procedures used to isolate and prepare the vector DNA for cloning • An explanation of the expected results for BOTH the cat and kan insert positive clones that were supposed to be plated onto antibiotic plates. Discuss which plates would and wouldn’t show growth for each respective insert. • Any applicable graphs, tables, and photos must be included in your typed report and should be labeled so that they can be referenced in the conclusion of the report. • Your report must contain the following data: o Calculations used in determining concentrations of DNA and how much DNA needed to be added to ligation reactions o An overview of the transformation procedure and the logic you would have used for plating the cloned transformants onto specific antibiotic plates and the expected results for each insert. Grading Rubric Typed Lab Report- (85 pts.) Proper Format of typed report– 5 pts. • 5 pts. – All sections are included and properly labeled. Headings for each section stand out. Overall appearance is nice and neat. • 3-4 pts. – Not all sections are clearly labeled. Overall appearance is somewhat disheveled. • 1-2 pts. – Sections are not labeled or are missing. Overall appearance is disheveled. • 0 pts. – None of the sections are labeled. Overall appearance is poor. Purpose/Relevance– 10 pts. • 10-8 pts. – The overall goal of the project is discussed and how it is applicable to the real-life scientific community • 7-5 pts. – The overall goal of the project is stated in the students’ own terms but the discussion of how the project is applicable to real-life projects is minimal. • 4-2 pts. – The purpose is copied from the manual and the relevance is missing. • 1-0 pts. – The purpose and relevance are missing. 68 IE #3 Lab report Analysis of Unknown Insert Mode #1 MCB 251 SP20 Procedure/Protocol– 10 pts. Procedures must be included for: • Restriction Digestion of a Vector • Clean up of Vector including all calculations • Transformation • 10-8 pts. – The procedure/protocol is well summarized and appears to be well understood; clear notation has been made of any and all modifications if necessary. • 7-5 pts. – The procedure/protocol is copied directly from the manual; modifications are carelessly noted. • 4-2 pt. – The procedure/protocol is copied from the manual incompletely; notation about modifications are missing • 1-0 pts. – The procedure/protocol is missing. Data/Results – 20 pts. Expected plate results for both kan and cat inserts on LA, LA + amp, LA + kan, LA + chlor, LA + amp + X-gal. Making a table of the results will suffice. • 20-17 pts. – All results are presented, clearly labeled and separated into sub-areas with high quality appearance. Including charts, graphs and/or drawings, and photos. • 17-14 pts. – All results are presented, clearly labeled and separated with a good quality appearance. Including charts, graphs and/or drawings, and photos. • 13-10 pts. – All results are presented, moderately labeled with a fair quality appearance. Including charts, graphs and/or drawings, and photos. • 9-6 pts. – Most results are presented, haphazardly labeled with a fair quality appearance. Including charts, graphs and/or drawings, and photos. • 5-2 pts. – Some results are presented, unlabeled with a poor appearance. • 1-0 pts. – Results are missing. Conclusion – 40 pts. You must discuss the following: • A molecular explanation for the observed growth on EACH plate. • The type of population is growing on each plate. • Discuss what information can be extracted from each particular plate. • 40-35 pts. – All results are clearly interpreted; hypotheses are made and evaluated for the obtained results. All experimental questions are answered thoughtfully and completely. Overall, the writing is concise, complete and of outstanding quality grammatically. There is a good synthesis of the experiment and the results, particularly in the scope of a larger context. • 34-29 pts. – All results are clearly interpreted; hypotheses are made and evaluated for the obtained results. All experimental questions are answered thoughtfully and completely. Overall, the writing is concise, complete and of high quality grammatically. The synthesis of the experiment and the results are limited to the context of the experiment. 69 IE #3 Lab report Analysis of Unknown Insert Mode #1 • • • • • MCB 251 SP20 28-23 pts. – Most results are addressed; experimental questions are not answered with well-formulated or complete answers. The writing is not concise or complete but is of good quality. The synthesis of the experiment and the results are not all relevant to the context of the experiment. 22-17 pts. – Less than half of results are addressed; experimental questions are not adequately answered. The writing is of fair quality. The synthesis of the experiment and the results are not at all relevant to the context of the experiment. 16-11 pts. – Minimal results are addressed; hypotheses for unexpected results are missing; experimental questions are not answered. The writing is of poor quality. Synthesizing thoughts about the experiment and its context are minimal. 10-4 pts. – Results are acknowledged; hypotheses are missing; experimental questions are unanswered. The writing is of poor quality and synthesizing thoughts are missing. 4-0 pts. – The conclusion is missing. 70 You are trying to clone the insert DNA into the vector plasmid. Here are the materials you were given: • Plasmid DNA (concentration = 150 ng/µl) • pBR322 • pBLU • pAMP • Insert DNA (concentrations = 5 ng/µl) • kan gene (kanamycin resistance gene) • cat gene (chloramphenicol resistance gene) Both DNA molecules must be digested with the same restriction enzyme to facilitate the ligation reaction. The insert DNA was already digested with BamHI prior to the beginning of this experiment. Therefore, last week, you digested the plasmid of choice with BamHI as well Clean-up to eliminate restriction enzyme Remember that restriction enzymes and ligases have opposing functions. This means that as long as active restriction enzymes are present, the ligase activity is going to be compromised. If the overall goal is to clone the insert DNA into the vector plasmid, we must eliminate the restriction enzymes. There are two ways to do so: 1. Heat the digestion reaction at 65-80°C for 20 minutes* 2. Get rid of the enzymes using a QIAGEN in silica column We will use the latter method for this class. *The temperature for heat inactivation depends on the restriction enzyme. General Clean Up Restriction Digestion Protocol(include in paper) 1. To each restriction reaction, add 250 µL buffer PB. a. Note that this is five times the volume of the digestion. If you have used a different volume for whatever reason, adjust this number accordingly. 2. Load entire volume to one of the QIAGEN columns in your kit. 3. Spin 30s at full speed in a microfuge. 4. Discard the effluent. 5. Add 750 µL buffer PE. 6. Spin 30s at full speed in a microfuge. 7. Discard the effluent. 8. Spin 30s at full speed in a microfuge. 9. Transfer the spin column (the blue insert with the white wafer in the bottom) to a fresh microfuge tube. 10. Add 30 µL EB. a. Be very careful to add this only to the center of the wafer. We are using 30 µL to reduce the overall recovery volume so as to increase the concentration. 11. Wait 1-2 minutes. Spin 30s at full speed in a microfuge. LIGATION Now you have both the vector plasmid and insert DNA that are digested with BamHI and cleaned. You are ready for ligation. Remember that we need a molar ratio of 3:1 (insert to vector). Note that the ratio is not by volume. This requires some calculations. Vector plasmid DNA: 5 µl of plasmid DNA (150 ng/µl) went into the digestion reaction (total volume of 20 µl). In another words, 5 µl of the total volume make up the plasmid DNA content. But during the clean up stage, you loaded all of the digested DNA onto the QIAGEN column and eluted it into 30 µl. When the volume changes, the concentration changes too. We use Vinitial x Cinitial = Vfinal x Cfinal where V = volume C = concentration Now you must calculate the necessary volume for the insert DNA. A couple of things to keep in mind when doing so. • The insert DNA is already digested and cleaned (essentially the same step as Step 1). The concentration is 5 ng/µl. • Based on the information found on the MCB 251 course website, kan is 1478 bp and cat is 1015 bp. Because you don’t know which insert you are working with, let’s approximate the insert DNA length by averaging the two values. Remember that you need 0.06 pmol of the insert, not 0.02 pmol Follow these protocols Transformation protocols (need to be included in paper) 1. Obtain 1 tubes of DH5 cells from the ice bucket on the backbench. Each tube contains 170 µl of competent cells. Label each respective microfuge tubes “P” for plasmid and “NP” for No Plasmid. Transfer 80 µl into each tube (P and NP) 2. Add 4 µl from the uncut purified plasmid at the back bench to the tube containing 80 µl of chemically competent cells labeled “P”. Add no DNA to the tube labeled “NP”. 3. Incubate on ice for 30 minutes. 4. Place both tubes in a floating microfuge tube rack in the 42ºC water bath at the back of the lab for 90 seconds. 5. Place both tubes on ice for 2 minutes. 6. Add 800 µl of SOC broth to each of the microfuge tubes. 7. Place both tubes in your section’s microfuge rack to be taken to the shaker/incubator. Prep Staff will take these and put them in the incubator. The cells will be grown up at 37ºC for 1 hour and then returned to the lab. 8. Using the technique for preparing a spread plate, inoculate a LB/ampicillin plate with 100 µl from each of the 2 cultures. Prepare the spread plates one at a time in order to prevent the cultures from drying out on the plates. Make sure you are labeling your plates with what culture each is being inoculated with! 9. When the plates are dry, place upside down on the trays at the back of the lab to be incubated. All plates will be incubated at 37ºC overnight and stored in the refrigerator until week 7. Restriction is a term used to describe the double-stranded cutting of DNA. Ligation (from the Latin ligare, “to tie”) is a term used to describe the ‘stitching’ or ‘tieing’ together of DNA strands. DNA ligases are natively used in cells to repair nicks and breaks in the phosphodiester backbone. Restriction digestion can result in so-called sticky ends that have overhangs as shown in the figure to the right. If two fragments of DNA have overhangs that are complementary, as in the figure, they can base pair and ligation can take place, fusing the fragments. Restriction digestion can result in so-called sticky ends that have overhangs as shown in the figure to the right. If two fragments of DNA have overhangs that are complementary, as in the figure, they can base pair and ligation can take place, fusing the fragments. Things to consider for implementing your experimental plans  How can you use restriction enzymes to clone your unknown insert into a vector?  If you digest DNA with restriction enzymes what do you think you would need to do in order to successfully ligate the digested DNA back together. Are the restriction enzymes still functional after a digestion? How could this affect your ligation efficiency?  What information do you need to set up a ligation reaction? Think about this in terms of how much vector DNA is needed and how much insert DNA is needed. What do you need to do in order to obtain this information? Think about how much digested vector DNA you will need. How can you obtain a larger volume of digested vector to work with?  Think about your experiment in the context of the vector concentration. How can you ensure the concentration of your vector is adequate after you clean up the plasmid to remove the restriction enzymes? Cloning – General Clean Up Restriction Digestion Protocol(include in paper) 1. To each restriction reaction, add 250 µL buffer PB. a. Note that this is five times the volume of the digestion. If you have used a different volume for whatever reason, adjust this number accordingly. 2. Load entire volume to one of the Qiagen columns in your kit. 3. 4. 5. 6. 7. 8. 9. Spin 30s at full speed in a microfuge. Discard the effluent. Add 750 µL buffer PE. Spin 30s at full speed in a microfuge. Discard the effluent. Spin 30s at full speed in a microfuge. Transfer the spin column (the blue insert with the white wafer in the bottom) to a fresh microfuge tube. 10. Add 30 µL EB. a. Be very careful to add this only to the center of the wafer. We are using 30 µL to reduce the overall recovery volume so as to increase the concentration. 11. Wait 1-2 minutes. Spin 30s at full speed in a microfuge