Team:WashU StLouis/Journal/charlotte.json
[
{
"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",
"-place sensor in a flask of water (woμLd leave sensor in holder for hard goods)"
]
}
]
},
{
"lead":"Make CμLtures",
"id":"C_exp2",
"goto":[
"C_exp3"
],
"description":"Make overnight cμLtures 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 cμLtures 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 cμLture (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":"-900 μL of H20 and 100 μL of JM109"},
{"what":"-900 μL of H20 and 100 μL of MG1655"},
{"what":"Create a blank with only water"}
]
},
{
"what":"Measure the OD600",
"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", "resμLt":"-Second measurement of OD of JM109: 0.375"},
{"what":"- In cold (4°C) room, evenly distribute each cμLture 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—shoμLd 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 cμLture tube"},
{"what":"- Place cμLture 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":"-Placed on shaker at 9:20 am – leave there until ~ 2 or 3 pm"},
{"what":"-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":"ShoμLd now be cloudy"},
{"what":"- Centrifuge at 4700 rpm for 15 minutes", "precaution":"-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":"-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":"-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 ug/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":"-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 μL.",
"id":"C_exp10",
"from":[
"C_exp9"
],
"steps":[
{"what":"Use 2 μL 10x buffer with green dye"},
{"what":"Enough volume of PCR product to provide 500 ng"},
{"what":"1 μL of each restriction enzyme (Xho1 and EcoRI)"},
{"what":"Water to reach 20 μL 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"
}
],
"resμLt":[
{
"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 .7μL buffer, 1 μL enzyme, .7 μL digested plasmid, 5x more insert DNA than plasmid DNA, water to 7 μL"},
{"what":"thermocyle under following conditions: 1) 37 degrees for 3 minutes, 2) 22 degrees for 3 minutes"}
]
},
{
"lead":"Overnight cμLtures",
"id":"C_exp14",
"from":[
"C_exp13"
],
"description":"Start an overnight cμLture 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 cμLture 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", "resμLt":"-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 cμLture 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":"-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/μL", "Take 2 μL of PE5A and 98 μL water", "Take 1 μL of that mixture and add 99 μL water", "Add 1 μL 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":[
"-Antibiotic is heat sensitive—wait until cooled on ice",
"-Antibiotic stocks in the lab are prepared such that 1 μL is needed per mL of agar",
"-Use chloramphenicol for the digests and negative control",
"-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":["-Plate 100 μL of that dilution on the amp plate",
"-Add sterile glass beads and shake laterally to spread around the cμLture",
"-Dump beads into nonsterile glass beads container"]
},
{"what":"- From centrifuged cμLtures, pipette off the media to the 100 μL mark and resuspend the pellet in that amount of media",
"steps":["-Add fμLl resuspended quantity to the correct labeled plate", "-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 CμLture",
"id":"C_exp20",
"description":"Start a cμLture 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 successfμLly 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":"First well undigested plasmid (2 μL 1:10 PA2C-TesA, 2 μL DNA loading dye 10x, and 16 μL water) -Second well: digested plasmid (3 μL 10x loading dye and digest product) -Last well: 1 kb plus ladder "
}
],
"resμLt":{
"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"
}
}
]
}
]