Difference between revisions of "Team:WashU StLouis/Journal/charlotte.json"
Ayekedavid (Talk | contribs) (Created page with " [ { "when":"6/2/15", "what":[ { "why":"We needed LB to grow this summer's cells", "lead":"Make Lb", "id":"C_exp1", "goto":[ ...") |
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{ | { | ||
"what":"add 2 L of water to a flask with 15 g yeast extract, 30 g NaCl, and 30 g tryptone", | "what":"add 2 L of water to a flask with 15 g yeast extract, 30 g NaCl, and 30 g tryptone", | ||
| − | "precaution":["Stir until disolved"] | + | "precaution":["Stir until disolved"] |
}, | }, | ||
{ | { | ||
| Line 62: | Line 62: | ||
"type":"P", | "type":"P", | ||
"steps":[ | "steps":[ | ||
| − | {"what":"• add 25 mL of each culture (JM109 and MG1655) into 500 mL of LB in a 2 L flask"} | + | {"what":"• add 25 mL of each culture (JM109 and MG1655) into 500 mL of LB in a 2 L flask"}, |
{"what":"• grow at 37°C and 250 rpm for ~1 hour", | {"what":"• grow at 37°C and 250 rpm for ~1 hour", | ||
"started":"10:33am", "ended":"11:35am"}, | "started":"10:33am", "ended":"11:35am"}, | ||
Revision as of 18:42, 26 June 2015
[
{
"when":"6/2/15",
"what":[
{
"why":"We needed LB to grow this summer's cells",
"lead":"Make Lb",
"id":"C_exp1",
"goto":[
"C_exp2"
],
"description":"We made LB Stock",
"type":"R",
"steps":[
{
"what":"add 2 L of water to a flask with 15 g yeast extract, 30 g NaCl, and 30 g tryptone",
"precaution":["Stir until disolved"]
},
{
"what":"add 500 mL of the dissolved solution to each of 3 bottles with 7.5 mL of agar"
},
{
"what":" autoclave the bottles",
"precaution":[
"loosen the lid of the bottles slightly",
"put autoclave tape crossing from the bottle onto the lid—can move to the lid later",
"o place sensor in a flask of water (would leave sensor in holder for hard goods)"
]
}
]
},
{
"lead":"Make Cultures",
"id":"C_exp2",
"goto":[
"C_exp3"
],
"description":"Make overnight cultures from frozen stock to make electrocompetent cells the next day (in Moon lab):",
"why":"The Moon Lab needed, strains JM109 and MG1655 for their experiments",
"type":"P",
"steps":[
{"what":"from glycerol stock, set up two 50mL cultures in 250-mL flasks"},
{"what":"grow overnight"}
]
}
]
},
{
"when":"6/3/15",
"what":[
{
"lead":"Competant Cell Prep",
"id":"C_exp3",
"goto":[
"C_exp4"
],
"description":"Electrocompetent prep of JM109 and MG1655 (in Moon lab):",
"why":"The Moon Lab needed, strains JM109 and MG1655 for their experiments",
"type":"P",
"steps":[
{"what":"• add 25 mL of each culture (JM109 and MG1655) into 500 mL of LB in a 2 L flask"},
{"what":"• grow at 37°C and 250 rpm for ~1 hour",
"started":"10:33am", "ended":"11:35am"},
{"what":"• add a 10x dilution into cuvettes",
"why":"The absorbance of light through these curvettes will determine whether enough cells have grown to continue the experiment",
"steps":[
{"what":"o 900 μL of H20 and 100 μL of JM109"},
{"what":"o 900 μL of H20 and 100 μL of MG1655"},
{"what":"Create a blank with only water"}
]
},
{"what":"Measure the OD600",
"result":"JM109 has OD .287, MG1655 has OD .390, the Desired range: 0.35 to 0.4. Because of the low absorbance we will let the cells incubate further",
"steps":[
{"what":" Put JM109 flask back in incubator for ~15 minutes starting at 11:56 am to try to increase OD"},
{"what":" Leave MG1655 on bench top (growing more slowly)"}
]
},
{"what":"Measure the OD600", "result":"o Second measurement of OD of JM109: 0.375"}
{"what":"• In cold (4°C) room, evenly distribute each culture into 2 centrifuge bottles (total of 4 bottles) and place in an ice bucket. Wait for 15-30 minutes.",
"started":"12:30 pm"
},
{"what":"• Keep on ice and bring bottles to centrifuge. Set at 4°C and 3000 rpm for 25 minutes"},
{"what":"• Return to cold room and discard supernatant by pouring quickly and gently "},
{"what":"• Suspend with total 500 mL of water (125 mL each bottle) and swirl until pellet disappears"},
{"what":"• Centrifuge at 4°C and 3000 rpm for 25 minutes"},
{"what":"• In cold room, discard liquid and suspend pellet gently (by swirling) with total 220 mL of 10% glycerol (55 mL each bottle) until you do not see pellet"},
{"what":"• Aliquot contents of each bottle into 4 15-mL conical tubes (16 tubes total)"},
{"what":"• Centrifuge at 4°C and 3000 rpm for 25 minutes"},
{"what":"• In cold room, pour off the liquid and pipette out the remaining liquid"},
{"what":"• Resuspend pellets in 1 mL glycerol in each tube, then combine into 1 tube per strain"},
{"what":"• Centrifuge at 4°C and 3000 rpm for 25 minutes"},
{"what":"• In cold room pour out liquid and pipette of remaining liquid"},
{"what":"• Add 1.5 mL of 10% glycerol to each tube and resuspend"},
{"what":"• Aliquot 40 μL each into small labeled vials and place in a labeled box in the -80°C freezer"}
]
},
{"lead":"Make Lb",
"id":"C_exp4",
"goto":[
"C_exp5"
],
"description":"Transformation (in Moon lab):",
"why":"The Moon Lab needed, strains JM109 and MG1655 for their experiments",
"type":"P",
"steps":[
{"what":"• Take MG1655 and WM1788 electrocompetent cells out of -80°C and place on ice"},
{"what":"• Take PSL2397 (plasmid) out of -80°C and place on ice"},
{"what":"• Add 2 μL of PSL2397 directly into MG1655 tube"},
{"what":"• Set pipette over 40 μL and draw up the MG1655 and PSL2397 mixture and place into electroporation cuvette "},
{"what":"• Tap cuvette to ensure cells are at bottom of cuvette—should check to see that there are no gaps—and place into electroporator; turn on"},
{"what":"• Immediately add 500 μL of LB into the cuvette then pour into culture tube"},
{"what":"• Place culture tubes of WM1788 and MG1655 into incubator for 1 hour "}
]
}
]
},
{
"when":"6/4/15",
"what":[
{
"lead":"Competant Cell Prep",
"id":"C_exp5",
"goto":[
"C_exp6"
],
"description":"Isolate plasmid (Zhang lab):",
"type":"P",
"steps":[
{"what":"• Add 50 mL of LB to conical 50 ml tube",
"precaution":"• Work within 30 cm of flame and flame lip of glass flask at opening and closing"},
{"what":"• Add 50 μL of chloramphenicol and mix "},
{"what":"• Pour into 125 mL flask"},
{"what":"• Add 1.5 mL of PA2C-TesA (cells with plasmid to be isolated) with a 1000 μL pipette"},
{"what":"• Place flask on shaker in warm room at 250 rpm (200 to 250 rpm in standard for E. coli)"
,"steps":[
{"what":"o Placed on shaker at 9:20 am – leave there until ~ 2 or 3 pm"},
{"what":"o Took out of warm room/off shaker at 2:12pm"}
]
},
{"what":"• Pour into a 50 mL conical tube"},
{"what":"• Centrifuge until supernatant is clear and then pour off supernatant"},
{"what":"• Add 2.5 mL of resuspension buffer and pipette in and out to resuspend until no pellet remains"},
{"what":"• Add 2.75 mL of lysis buffer and invert a few times to mix", "precaution":"Solution must now be clear"},
{"what":"• Add 4 mL of neutralization buffer and invert a few times to mix", "precaution":"Should now be cloudy"},
{"what":"• Centrifuge at 4700 rpm for 15 minutes", "precaution":"o Supernatant not completely clear after centrifuging, but that is fine because it will be filtered"},
{"what":"• Filter into a new conical tube using a syringe with a cotton ball in it", "precaution":"o Filtered liquid is clear"},
{"what":"• Add 2.5 times the volume of the liquid of cold pure ethanol (~10 mL ~35 mL total volume"},
{"what":"• Put in -20°C for 20 minutes", "started":"4:00pm", "ended":"4:22pm"},
{"what":"• Centrifuge for 25 minutes at 4°C at 4700 rpm, then check for a pellet"},
{"what":"• Pour off supernatant", "precaution":"o Can tap the top of the tube against a paper towel to remove more ethanol"},
{"what":"• Add at least 20 mL of 70% EtOH and shake to break up the pellet, then fortex"},
{"what":"• Centrifuge at 3000 rpm for 7 minutes at 4°C"},
{"what":"• Pour off the supernatant"},
{"what":"• Resuspend in 500 μL of TE RNAse A (20 μg/mL)"},
{"what":"• Add 5 times that volume (2.5 mL) of BNL buffer (from old miniprep kit) and pipette in and out to mix"},
{"what":"• Add 750 μL of the solution to a spin mini column and collection tube"},
{"what":"• Balance centrifuge and spin for 1 minute at 12500 rpm; discard flow-through"},
{"what":"• Repeat with additional 750 μL until all of the solution has run through the column (centrifuge each time on the same settings and in the same column)"},
{"what":"• Spin down for 2 minutes at 12500 rpm to get rid of the rest of ethanol", "precaution":"o Optional step that was not performed: can put in 50°C room for a few minutes to evaporate the rest of the ethanol"},
{"what":"• Add 750 μL of wash buffer; spin and discard the flow-through"},
{"what":"• Repeat with additional 750 μL of wash buffer"},
{"what":"• Place column into a new Eppendorf tube and place 40 μL off eluent (in this case, water) directly onto the bottom of the column without touching it with the pipette tip"},
{"what":"• Wait ~3 minutes"},
{"what":"• Centrifuge at 13000 rpm for 2 minutes and label flow-through vial"},
{"what":"• Take measurement on nanodrop and label the vial with the concentration"}
]
},
{
"lead":"Make Lb",
"id":"C_exp6",
"description":"Use SnapGene to design primers for the 14 sequences to be overexpressed",
"type":"D",
"why":"There are 10 genes in the Nif cluster that have unknown functions. Overexpressing these can shead light on what they do",
"steps":[
{"what":"• Check for EcoRI and XhoI restriction sites within each of the sequences"},
{"what":"• Check for directionality on the plasmid: direct or complementary"},
{"what":"• If a restriction enzyme does not have sites within the sequence, add a site for that restriction enzyme to the appropriate primer and add 6 adenines beyond the site on the primer so that the restriction enzyme will work properly"},
{"what":"• If the restriction enzyme does have a site within the sequence, end the primer at the end of the sequence to be amplified to leave the ends blunt"},
{"what":"• If direct, add EcoRI to the 5’ end and XhoI to the 3’ end as appropriate"},
{"what":"• If complementary, add XhoI to the 5’ end and EcoRI to the 3’ end as appropriate"},
{"what":"• Maintain a Tm for each primer above 60°C and approximately match the Tm of paired primers"},
{"what":"• Ensure that there is only 1 binding site on the plasmid for each primer"}
]
}
]
},
{
"when":"6/9/2015",
"what":[
{
"lead":"Resuspend Primers",
"id":"C_exp7",
"description":"Resuspend the dry primers in Tris-EDTA (TE) solution",
"type":"P"},
{"lead":"Run PCR",
"id":"C_exp8",
"from":[
"C_exp7"
],
"description":"Run PCR on 3 of the 14 amplicons",
"extra":"Amplify hesA, nifB, and nifV",
"type":"P",
"pics":[
{
"src":"http://placehold.it/350x150",
"cap":"PCR Gel from 6/9/2015"
}
]
}
]
},
{
"when":"6/10/2015",
"What":[
{
"lead":"Run PCR",
"id":"C_exp9",
"from":[
"C_exp7"
],
"group":"C_exp8",
"description":"Run PCR on remaining amplicons", "type":"P",
"pics":[
{
"src":"http://placehold.it/350x150",
"cap":"PCR Gel from 6/10/2015"
}
]
}
]
},
{
"when":"6/11/2015",
"what":[
{
"lead":"Restriction Digest",
"description":"Perform a restriction enzyme digest of PCR products and of plasmid backbone using 20 ul.",
"id":"C_exp10",
"from":[
"C_exp9"
],
"steps":[
{"what":"Use 2 ul 10x buffer with green dye"},
{"what":"Enough volume of PCR product to provide 500 ng"},
{"what":"1 ul of each restriction enzyme (Xho1 and EcoRI)"},
{"what":"Water to reach 20 ul total volume"},
{"what":"Centrifuge for 1 min; incubate at 37 degrees for ~ 3 hrs"},
{"what":"Purify restriction digest products",
"steps":[
{"what":"Use Zymo Plasmid purification kit to purify digest products"}
]
},
{"what":"Measure DNA concentration with nanodrop"}
]
},
{
"lead":"Run PCR",
"id":"C_exp11",
"from":[
"C_exp10"
],
"description":"Run PCR on restriction digest products", "type":"P",
"pics":[
{
"src":"http://placehold.it/350x150",
"cap":"PCR Gel from 6/11/2015"
}
],
"result":[
{
"what":"Plasmid: Saw bands for fragment, backbone with fragment caught out, and linearized fragment. We hoped to only see the first two."
}
]
},
{
"lead":"Gel Purification",
"id":"C_exp12",
"from":[
"C_exp11"
],
"description":"Gel purify the backbone with fragment cut out from plasmid as well as the PCR product"
},
{
"lead":"Ligate products",
"id":"C_exp13",
"from":[
"C_exp12"
],
"description":"Set up 7 μL ligation reactions for the digestion products that were digested by both enzymes: (EcoR1 and Xho1)"
,"extra":"Set up an addition ligation reaction for negative control",
"steps":[
{"what":"Add .7ul buffer, 1 ul enzyme, .7 ul digested plasmid, 5x more insert DNA than plasmid DNA, water to 7 ul"},
{"what":"thermocyle under following conditions: 1) 37 degrees for 3 minutes, 2) 22 degrees for 3 minutes"}
]
},
{
"lead":"Overnight cultures",
"id":"C_exp14",
"from":[
"C_exp13"
],
"description":"Start an overnight culture in 5 mL of LB at 37°C for transformation of ligated plasmids"
}
]
},
{
"when":"6/12/2015",
"what":[
{
"lead":"Competent cell prep",
"id":"C_exp15",
"description":"Prepare competent cellls using the RbCl method",
"from":[
"C_exp14"
],
"steps":[
{"what":"• Dilute 1 mL of culture into 50 mL of LBMg medium pre-warmed to 37°C"},
{"what":"• Grow at 37°C for approximately 2 hours on shaker to OD600 of about 0.6", "result":"o Final OD600 = 0.598", "precaution":"• Do not vortex cells after this point or allow them to warm above 4°C"},
{"what":"• Incubate for 20 minutes on ice"},
{"what":"Transfer culture to an ice-cold 50 mL conical tube"},
{"what":"• Centrifuge for 15 minutes at 3000 rpm and 4°C; pour of the supernatant"},
{"what":"• Resuspend in 20 mL of Tbf1 from 4°C fridge"},
{"what":"• Incubate on ice for 25 minutes "},
{"what":"• Centrifuge for 10 minutes at 3000 rpm and 4°C; remove the supernatant"},
{"what":"• Resuspend in 4 mL of Tbf2 from the fridge"},
{"what":"• Aliquot 100 μL into microcentrifuge tubes", "precaution":"o Work in the hood with cells on ice and place filled tubes into liquid nitrogen"},
]
},
{
"lead":"Transform",
"id":"C_exp16",
"description":"Transform products from yesterday's ligation reaction into the cells",
"from":[
"C_exp15",
"C_exp13"
],
"steps":[
{"what":"Use PE5A plasmid ( with amplicilin resistance) as an additional control",
"steps":["Original concentration of plasmid: 375 ng/ul", "Take 2 ul of PE5A and 98 uL water", "Take 1 ul of that mixture and add 99 uL water", "Add 1 uL of that to a vial of competant cells"]},
"• Add the ligation reaction products to other labeled vials of competent cells ",
"• Heat shock at 42°C for 1 minute; replace on ice for ~2 minutes",
"• Add 900 μL of LB to each tube (working within the radius of flame)",
"• Place on shaker at 37°C for 1 hour"
]
},
{
"lead":"Make Plates",
"id":"C_exp17",
"group":"C_exp16",
"steps":[
"• Melt LB agar in the microwave; incubate on ice for 25 minutes",
{"what": "Add appropriate antibiotic to LB agar",
"steps":[
"o Antibiotic is heat sensitive—wait until cooled on ice",
"o Antibiotic stocks in the lab are prepared such that 1 μL is needed per mL of agar",
"o Use chloramphenicol for the digests and negative control",
"o Use ampicillin for the PE5A plasmid"
]
},
"• Pour ~20 mL of LB agar with antibiotic per plate; pour slowly to avoid bubbles",
"• Place in hood with lids slightly off to avoid condensation",
"• Label bottom of plates with antibiotic, gene, and date"
]
},
{
"lead":"Plate Cells",
"id":"C_exp18",
"group":"C_exp17",
"steps":[
"• Take cells out of 37°C room and spin down all but the control for efficiency of transformation (PE5A) for 4 minutes",
{"what":"• Take 100 μL from PE5A control and add to 900 μL of LB",
"steps":["o Plate 100 μL of that dilution on the amp plate",
"o Add sterile glass beads and shake laterally to spread around the culture",
"o Dump beads into nonsterile glass beads container"]
},
{"what":"• From centrifuged cultures, pipette off the media to the 100 μL mark and resuspend the pellet in that amount of media",
"steps":["o Add full resuspended quantity to the correct labeled plate", "o Add beads and shake"]},
"• Place all plates in 37°C room"
]
},
]
},{
"when":"6/13/15",
"what":[
{
"lead":"Store plates",
"id":"C_exp19",
"description":"Take plates out of 37°C room, check for colonies, and place those that grew into the 4°C room for the remainder of the weekend",
"from":"C_exp18"
}
]
},
{
"when":"6/15/15",
"what":[
{
"lead":"Start a Culture",
"id":"C_exp20",
"description":"Start a culture of PA2C-TesA in LB for ~4 hours"
},
{
"lead":"Redo PCR",
"id":"C_exp21",
"description":"Redo PCR using same conditions as previously for the genes that were not successfully transformed",
"from":"C_exp9"
},{
"lead":"Redo Purification",
"id":"C_exp22",
"description":"Purify the PCR product with a kit, as previously and take concentrations on nanodrop",
"group":"C_exp21"
},
{
"lead":"Redo Purification",
"id":"C_exp23",
"description":"Purify the PA2C-TesA plasmid as previously and take concentration on nanodrop",
"group":"C_exp21"
},
{
"lead":"Redo Digest",
"id":"C_exp24",
"description":"Set up 20 μL digest reactions for the newly purified genes using the same specifications as previously ",
"precaution":"Place at 37 degrees for ~ 2 hours"
},
{
"lead":"Run Gel",
"id":"C_exp25",
"description":"• Run digested plasmid through the gel ",
"pics":[
{"src":"http://placehold.it/350x150",
"caption":"o First well: undigested plasmid (2 μL 1:10 PA2C-TesA, 2 μL DNA loading dye 10x, and 16 μL water)
o Second well: digested plasmid (3 μL 10x loading dye and digest product)
o Last well: 1 kb plus ladder
"
}
],
"result":{
"what":"• Saw no band for digested plasmid and a band in unexpected location for the undigested plasmid need to redo the digestion of the plasmid"
}
}
]
}
]