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"}ol{margin:0;padding:0}table td,table th{padding:0}.c1{margin-left:72pt;orphans:2;widows:2;padding-left:0pt}.c2{margin-left:36pt;orphans:2;widows:2;padding-left:0pt}.c5{background-color:#ffffff;max-width:468pt;padding:72pt 72pt 72pt 72pt}.c3{orphans:2;widows:2}.c0{background-color:#ffffff;font-family:"Times New Roman"}.c6{padding:0;margin:0}.c11{margin-left:108pt;padding-left:0pt}.c4{font-weight:700;font-family:"Times New Roman"}.c10{margin-left:36pt}.c7{height:11pt}.c9{font-family:"Times New Roman"}.c8{font-weight:700}.title{padding-top:0pt;color:#000000;font-size:26pt;padding-bottom:3pt;font-family:"Arial";line-height:1.15;page-break-after:avoid;orphans:2;widows:2;text-align:left}.subtitle{padding-top:0pt;color:#666666;font-size:15pt;padding-bottom:16pt;font-family:"Arial";line-height:1.15;page-break-after:avoid;orphans:2;widows:2;text-align:left}li{color:#000000;font-size:11pt;font-family:"Arial"}p{margin:0;color:#000000;font-size:11pt;font-family:"Arial"}h1{padding-top:20pt;color:#000000;font-size:20pt;padding-bottom:6pt;font-family:"Arial";line-height:1.15;page-break-after:avoid;orphans:2;widows:2;text-align:left}h2{padding-top:18pt;color:#000000;font-size:16pt;padding-bottom:6pt;font-family:"Arial";line-height:1.15;page-break-after:avoid;orphans:2;widows:2;text-align:left}h3{padding-top:16pt;color:#434343;font-size:14pt;padding-bottom:4pt;font-family:"Arial";line-height:1.15;page-break-after:avoid;orphans:2;widows:2;text-align:left}h4{padding-top:14pt;color:#666666;font-size:12pt;padding-bottom:4pt;font-family:"Arial";line-height:1.15;page-break-after:avoid;orphans:2;widows:2;text-align:left}h5{padding-top:12pt;color:#666666;font-size:11pt;padding-bottom:4pt;font-family:"Arial";line-height:1.15;page-break-after:avoid;orphans:2;widows:2;text-align:left}h6{padding-top:12pt;color:#666666;font-size:11pt;padding-bottom:4pt;font-family:"Arial";line-height:1.15;page-break-after:avoid;font-style:italic;orphans:2;widows:2;text-align:left}</style></head><body class="c5"><p class="c3"><span class="c4">Day 0</span><p class="c3"><span class="c9">Streak out frozen stocks of two strains, which consist of a biobrick (K592009), vector (K608002), and type of</span><span class="c9"> E. coli</span><span class="c9"> (TOP10 and MDS42), on separate plates.</span><ol class="c6 lst-kix_hlvzwhmd082q-0 start" start="1"><li class="c2"><span class="c9">Find two LB/CAM plates from the cold room (</span><span class="c0">4°C)</span><span class="c9"> and a bench with a Bunsen burner</span></li><li class="c2"><span class="c9">Get ice bucket and fill with ice</span></li><li class="c2"><span class="c9">Collect previously made frozen glycerol stocks (TOP10 and MDS42) from the -80</span><span class="c0">°C freezer and place them on ice</span></li><li class="c2"><span class="c0">Quickly return to your bench and demonstrate sterile technique (sterilize bench top with 70% EtOH and then light your bunsen burner)</span></li><li class="c2"><span class="c0">Sterilize an inoculation loop (immerse in 70% EtOH and flame until the loop is visibly red hot)</span></li><li class="c2"><span class="c0">Use the inoculation loop to streak out your cells onto each plate (refer to Lab 2 reading streaking procedure). Flame in between streakings.</span></li><li class="c2"><span class="c0">Flip the plates upside down and place them in the 37°C incubator overnight</span></li></ol><p class="c3 c7"><span class="c0"></span><p class="c3"><span class="c0 c8">Day 1</span><p class="c3"><span class="c0">Your plates will need to grow in the incubator for at least 20 hours. </span><p class="c3"><span class="c0">Record/document observations of your plates. How many colonies? Colors? Are they isolated? Anything interesting? (take pictures)</span><p class="c3"><span class="c0">Pick six independent colonies from each plate that appear to maintain the desired phenotype and start six 5 ml overnight cultures in LB/CAM media for each strain. </span><ol class="c6 lst-kix_sirnd5wr6i6g-0 start" start="1"><li class="c2"><span class="c0">Sterilize your bench and turn on the Bunsen burner</span></li><li class="c2"><span class="c0">Collect 12 culture tubes and label them (Day 1, your initials, date, strain, and colony #1-6)</span></li><li class="c2"><span class="c0">Fill each culture tube with 5ml of LB/CAM culture using a disposable glass pipette</span></li><li class="c2"><span class="c0">If your protein has a distinct color, choose colonies that visibly show that color. This is not guaranteed for all devices, since very unstable devices may already be broken. If there aren’t enough colonies with the desired phenotype, make a new plate. </span></li><li class="c2"><span class="c0">Circle 6 chosen colonies with a sharpie on each plate</span></li><li class="c2"><span class="c0">How to pick the colonies:</span></li></ol><ol class="c6 lst-kix_sirnd5wr6i6g-1 start" start="1"><li class="c1"><span class="c0">Using a sterile p1000 tip, gently pierce through the entire colony and agar</span></li><li class="c1"><span class="c0">Place the entire tip into the culture tube. Slowly and gently pipette up and down to dislodge the agar and colony</span></li><li class="c1"><span class="c0">Then, gently eject the tip so that it remains in the culture tube</span></li><li class="c1"><span class="c0">Do this for all 6 chosen colonies</span></li></ol><ol class="c6 lst-kix_sirnd5wr6i6g-0" start="7"><li class="c2"><span class="c0">Repeat this for both strains</span></li><li class="c2"><span class="c0">Place all culture tubes into the shaker incubator (37°C and at 200-225 RPM) overnight</span></li><li class="c2"><span class="c0">Place the plates in the cold room (4°C) on the shelf labeled for your group</span></li></ol><p class="c3 c7"><span class="c0 c8"></span><p class="c3"><span class="c0 c8">Day 2</span><p class="c3"><span class="c0">Your cultures will need to grow in the incubator for at least 20 hours. </span><p class="c3"><span class="c0">Record/document observations of your cultures. Color? Cloudy? Translucent? How many generations have passed? (take pictures)</span><p class="c3"><span class="c0">Perform a 1:1000 dilution using LB/CAM media on each of your overnight cultures so that the growth starts once more from the first generation. </span><ol class="c6 lst-kix_wk0a504iaux6-0 start" start="1"><li class="c2"><span class="c0">Turn on the Bunsen burner and use sterile technique</span></li><li class="c2"><span class="c0">Find 12 microcentrifuge tubes and label them (1 for each O/N culture-strain and colony #)</span></li><li class="c2"><span class="c0">Make sure the cultures are fully resuspended. If they were sitting in the cold room or a bench for more than 15 minutes, carefully vortex the culture until a homogenous mixture is created</span></li><li class="c2"><span class="c0">Remove 250 µl from each overnight culture and place into its respective microcentrifuge tube</span></li><li class="c2"><span class="c0">Start a fresh 5 ml culture for each O/N culture</span></li></ol><ol class="c6 lst-kix_wk0a504iaux6-1 start" start="1"><li class="c1"><span class="c0">Locate 12 new culture tubes and label them (Day 2, your initials, date, strain, and colony #1-6)</span></li><li class="c1"><span class="c0">Pipette 5 ml of LB/CAM media into each culture tube using the glass pipette</span></li><li class="c1"><span class="c0">You will be performing a 1:1000 dilution of the “Day 1” culture into the new media</span></li><li class="c1"><span class="c0">Add 5 µl of each “Day 1” culture from its microcentrifuge tube into its respective new culture tube</span></li><li class="c1"><span class="c0">Place these “Day 2” cultures back into the shaker incubator (37°C and at 200-225 RPM) overnight</span></li></ol><ol class="c6 lst-kix_wk0a504iaux6-0" start="6"><li class="c2"><span class="c0">Measure the chromoprotein absorbance for your cultures (intended color)</span></li></ol><ol class="c6 lst-kix_wk0a504iaux6-1 start" start="1"><li class="c1"><span class="c0">From the remaining culture in the microcentrifuge tube, add 200 µl to a well in a 96-well plate. There will be a single plate being used each day (ask Dr. Mishler)</span></li><li class="c1"><span class="c0">Record what wells you will be pipetting into, and carefully add the 200 µl to just those wells. Avoid cross contamination. </span></li><li class="c1"><span class="c0">Return the plate to a mentor/Dr. Mishler, who will store it in the cold room</span></li><li class="c1"><span class="c0">The plate will be read by Dr. Mishler or a mentor later, once all of the samples for the day have been added</span></li></ol><ol class="c6 lst-kix_wk0a504iaux6-0" start="7"><li class="c2"><span class="c0">Clean up your bench space and store your overnight cultures from the previous day in the cold room on your group’s shelf</span></li></ol><p class="c3 c7"><span class="c0 c8"></span><p class="c3"><span class="c0 c8">Day 3 → until completion</span><p class="c3"><span class="c0">Repeat the procedure for Day 2, preparing new culture tubes with labeled with the respective days. </span><p class="c3"><span class="c0">Record the absorbance readings from the previous day into your notebook, if they are available. This should be accessible online in our google drive folder. </span><p class="c3"><span class="c0">Record how many generations passed between each day.</span><p class="c3 c7"><span class="c0"></span><p class="c3"><span class="c0">After 7-10+ days, or after a time when the color of the cultures visibly diminishes, you will be asked to stop carrying forward the overnight cultures. You will want to check this with Dr. Mishler or a mentor. In general, we will want to see that the color has declined and stayed declined for at least 2 consecutive days. </span><p class="c3 c7"><span class="c0"></span><p class="c3"><span class="c0 c8">Day “completion” = Day X</span><p class="c3"><span class="c0">In consultation with Dr. Mishler, stop growing cultures once the color has significantly diminished. </span><ol class="c6 lst-kix_j6e0b0djltj7-0 start" start="1"><li class="c2"><span class="c0">Plate a dilution of each O/N culture to generate single colonies</span></li></ol><ol class="c6 lst-kix_j6e0b0djltj7-1 start" start="1"><li class="c1"><span class="c0">First, find 12 LB/CAM agar plates from the cold room. If there are none, then make some for your group. </span></li><li class="c1"><span class="c0">Turn on the Bunsen burner and use sterile technique</span></li><li class="c1"><span class="c0">Get 12 empty culture tubes (1 for each O/N culture) and label them according to their respective strain and colony #</span></li><li class="c1"><span class="c0">Fill each of them with 5 ml of fresh LB/CAM broth using the glass pipette</span></li><li class="c1"><span class="c0">Add 5 µl of each O/N culture to its respective new culture tube. This is a 1:1000 dilution. </span></li><li class="c1"><span class="c0">Then, briefly vortex the cultures and let them sit for 1 minute. </span></li><li class="c1"><span class="c0">Get 12 more empty culture tubes (1 for each culture) and label them once more, according to their respective strain and colony #</span></li><li class="c1"><span class="c0">Fill each of them with 5 ml of fresh LB/CAM broth using the glass pipette</span></li><li class="c1"><span class="c0">Add 5 µl of the 1:1000 dilution culture to its respective new culture tube. This is a 1:10^6 or a “1 to a million” dilution</span></li><li class="c1"><span class="c0">Again, briefly vortex the cultures and let them sit for 1 minute</span></li><li class="c1"><span class="c0">Label each LB/CAM plate with your initials, strain name, colony number, the date, and the day # of your culture tube</span></li><li class="c1"><span class="c0">Plate 50 µl of the 1:10^6 dilutions onto an LB/CAM agar plate using a sterilized spreader. Do this for all 12 dilutions. </span></li><li class="c1"><span class="c0">Place the plates into the 37°C incubator overnight</span></li></ol><ol class="c6 lst-kix_j6e0b0djltj7-0" start="2"><li class="c2"><span class="c0">Prepare a glycerol stock for each final culture</span></li></ol><ol class="c6 lst-kix_j6e0b0djltj7-1 start" start="1"><li class="c1"><span class="c0">Find 12 “cryo vials”</span></li><li class="c1"><span class="c0">Label the lid of the vial with your initials, “BiB”, and the strain number (not name)</span></li><li class="c1"><span class="c0">Label the side of the vial (white portion) with your initials, the strain name and colony #, the date, and the day # of the culture</span></li><li class="c1"><span class="c0">Using sterile conditions, make glycerol stocks for each culture, the procedure for which can be found in the procedure manual. </span></li><li class="c1"><span class="c0">Mix the glycerol stocks via vortexing/vigorous inverting</span></li><li class="c1"><span class="c0">Place these stocks in the -80°C freezer, in a designated box</span></li></ol><ol class="c6 lst-kix_j6e0b0djltj7-0" start="3"><li class="c2"><span class="c0">Final absorbance readings</span></li></ol><ol class="c6 lst-kix_j6e0b0djltj7-1 start" start="1"><li class="c1"><span class="c0">Using your saturated O/N cultures, prepare a final set of of absorbance readings. Add your cultures to the 96-well plate as you previously have done</span></li></ol><ol class="c6 lst-kix_j6e0b0djltj7-0" start="4"><li class="c2"><span class="c0">Take a picture of your cultures from Day 1→ today</span></li></ol><ol class="c6 lst-kix_j6e0b0djltj7-1 start" start="1"><li class="c1"><span class="c0">Find your cultures in the cold room</span></li><li class="c1"><span class="c0">Arrange these cultures in day order and a take a picture for all 6 sets for each strain</span></li></ol><p class="c3 c7"><span class="c0 c8"></span><p class="c3"><span class="c0 c8">Day X+1</span><ol class="c6 lst-kix_fi84u8xe091x-0 start" start="1"><li class="c2"><span class="c0">Remove your plates from the incubator and take a picture of them side by side with your original day 0 plate (find in cold room)</span></li><li class="c2"><span class="c0">Record your observations of the plates in your lab notebook</span></li><li class="c2"><span class="c0">Pick 1 colony from each of your 12 plates</span></li></ol><ol class="c6 lst-kix_fi84u8xe091x-1 start" start="1"><li class="c1"><span class="c0">This colony should not be strongly colored</span></li><li class="c1"><span class="c0">This colony should be representative of the majority of colonies on the plate</span></li><li class="c1"><span class="c0">Pick a random colony, and mark each chosen colony with a sharpie on the back of the plate</span></li></ol><ol class="c6 lst-kix_fi84u8xe091x-0" start="4"><li class="c2"><span class="c0">Turn on a Bunsen burner and use sterile technique</span></li><li class="c2"><span class="c0">Locate 12 culture tubes label them for each strain and colony #</span></li><li class="c2"><span class="c0">Fill them each with 5 ml of LB/CAM broth using a glass pipette</span></li><li class="c2"><span class="c0">Gently touch each chosen colony with a red pipette tip and drop the tip into its respective culture tube</span></li><li class="c2"><span class="c0">Place these tubes in the shaker incubator (37°C and at 200-225 RPM) overnight</span></li></ol><p class="c3 c7 c10"><span class="c0"></span><p class="c3"><span class="c0 c8">Day X+2</span><ol class="c6 lst-kix_z85vgyq93hwo-0 start" start="1"><li class="c2"><span class="c0">Prepare a glycerol stock for each colony culture</span></li></ol><ol class="c6 lst-kix_z85vgyq93hwo-1 start" start="1"><li class="c1"><span class="c0">Find 12 “cryo vials”</span></li><li class="c1"><span class="c0">Label the lid of the vial with your initials, BiB, strain number followed by “C” (the “C” stands for colony)</span></li><li class="c1"><span class="c0">Label the side of the vial (white portion) with your initials, the strain name, colony number, the date, the day # of the culture, and “colony”</span></li><li class="c1"><span class="c0">Make the glycerol stock using the previously described procedure</span></li><li class="c1"><span class="c0">Mix via vortexing/vigorous inverting</span></li><li class="c1"><span class="c0">Store in the -80°C freezer, in a designated box</span></li></ol><ol class="c6 lst-kix_z85vgyq93hwo-0" start="2"><li class="c2"><span class="c0">Miniprep each O/N culture and send them out for sequencing</span></li></ol><ol class="c6 lst-kix_z85vgyq93hwo-1 start" start="1"><li class="c1"><span class="c0">Miniprep the six O/N cultures using the procedure given in the miniprep kit</span></li><li class="c1"><span class="c0">Before you begin the manual procedure, you will need to vortex the culture</span></li><li class="c1"><span class="c0">Upon completing the miniprep process, you will elute into a fresh microcentrifuge tube. Label each:</span></li></ol><ol class="c6 lst-kix_z85vgyq93hwo-2 start" start="1"><li class="c3 c11"><span class="c0">On lid- “Mini”, your initials, BiB, and strain number followed by “C”</span></li><li class="c3 c11"><span class="c0">On side- your initials, the date, culture #, and day #</span></li></ol><ol class="c6 lst-kix_z85vgyq93hwo-1" start="4"><li class="c1"><span class="c0">Nanodrop these samples and record the concentrations on the side of the tube and in your notebook. </span></li><li class="c1"><span class="c0">Prepare the samples for sequencing according to their concentrations. Follow the procedure given in the Lab Procedures/Protocols manual</span></li></ol></body></html> -- Main.SeanLeonard - 25 Aug 2016
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