Difference between revisions of "Team:US AFRL CarrollHS/Experiments"

<h2>Transformations</h2>

<h3>BI21 Cells:</h3>

<li>Thaw 50 µL vial of BL21 cells on ice</li>

<li>Inoculate 2µL DNA into 50µL BL21 cells</li>

<li>incubate on ice for 30 minutes</li>

<li>heat shock at 42℃ for 10 seconds</li>

<li>incubate on ice for 5m</li>

<li>Inoculate 250µL LB into vial</li>

<li>incubate at 37℃ with shaking for 1.5 hours</li>

<li>Plate 1x/9x</li>

<h3>JM109 Cells:</h3>

<li>Thaw vial of JM109 cells on ice</li>

<li>Label 14 mL Falcon tube & plane on ice</li>

<li>Inoculate 50 µL JM109 cells into Falcon tube</li>

<li>Inoculate 2µL DNA into 50µL JM109 cells</li>

<li>incubate on ice for 20m</li>

<li>heat shock at 42℃ for 45 seconds</li>

<li>Inoculate 950µL LB into vial</li>

<li>incubate at 37℃ with shaking for 1.5 hours</li>

<li>Plate 1x/9x</li>

<h2>Electrophoresis/Gel Protocols</h2>

<h3>1% Agarose Electrophoresis Gel</h3>

<li>Add 0.35 g agarose to 35 mL 1x TAE in 100mL conical flask</li>

<li>Heat in microwave for 35 seconds</li>

<li>Swirl and heat in microwave for 25 seconds</li>

<li>Swirl and heat in microwave for 15 seconds</li>

<li>Swirl and heat in microwave for 7 seconds</li>

<li>Swirl and ensure no ‘floaties’ </li>

<li>Cool outside of conical flask with water until ‘hand not’</li>

<li>Pour into gel mold and add well comb</li>

<h3>Gel Extraction</h3>

<li>Use ethanol-wiped scalpel to excise appropriate bands</li>

<li>Mix and incubate at 50°C for 10 min</li>

<li>Vortex every 2 mins until gel slice is completely dissolved</li>

<li>Place nucleospin column into 2mL collection tube</li>

<li>Add sample</li>

<li>Centrifuge at 11,000 xg for 1 minute</li>

<li>Discard throughflow and place column back in same tube</li>

<li>Add 700 mL Buffer NT3</li>

<li>Centrifuge at 11,000 xg for 1 minute</li>

<li>Discard throughflow and place column back in same tube</li>

<li>Repeat step 4</li>

<li>Centrifuge at 11,000 xg for another minute</li>

<li>To remove buffer</li>

<li>Discard throughflow</li>

<li>Centrifuge at 11,00 xg for add minute</li>

<li>Place column in a clean, labelled 1.5 mL tube </li>

<li>Add 25 mL prewarmed (50°C) Buffer NE</li>

<li>Incubate at 50°C for 5 min</li>

<li>Centrifuge at 50 xg for 1 minute</li>

<li>Centrifuge at 11,000 xg for 1 minute</li>

<h2>Plasmid Transformation Protocol</h2>

<li>Aliquot 1 ml media into 1.5 mL tubes</li>

    <li>Place in 42°C waterbath</li>

<li>Prechill labelled 14 mL round-bottom falcon tubes</li>

  <li>Add mL cell culture to tube</li>

  <li>Add appropriate amount of ligase rxh (2-2.5 L)</li>  

  <li>Incubate in nice for 20 minutes</li>

<li>Heat shock cells by placing at 42℃ for 45 seconds in waterbath </li>

  <li>Place on ice for 2 minutes</li>

<li>Add 950 mL preheated media to each tube</li>

<li>Incubate at 37℃ with 215 rpm shaking for 1.5 hr -> 20 minutes</li>

<h3>Plating</h3>

<li>Plate on LB-antibiotic plates</li>

<li>Plate 100 ml for 1x transformation</li>  

<li>Plate 100 mL 9x centrifuge remaining transformation at 2000 xg for 5 minutes and resuspended pellet in 100 L media </li>

<h2>Plates Protocol</h2>

<li>Follow LB Agar Recipe </li>

  <li>Make sure to put stirring bar in water</li>

  <li>Needed to make into homogenous gel</li>

  <li>Needs to be sterile</li>

<li>Put into Autoclave </li>

  <li>Lid should not be too tight</li>

<li>Put in ice bath42°C</li>

  <li>Cool till not burning hand</li>

  <li>Put on stirring plate</li>

<li>Put sterilized plates in Lamenia shield</li>

<li>Using micropipette put .5 mL of chlor. And amp. agar solution</li>

  <li>1000x dilution</li>

  <li>Amp. has to be defrosted in dark drawer</li>

<li>Pour solution into plates</li>

  <li>Thin layer fill bottom</li>

  <li>Take lid slightly off to prevent condensation</li>

<h2>QIA Prep Spin Mini Prep Kit</h2>

<li>Example for 18 mL LB add</li>

  <li>18 L20 mg/ L Kanamycin (antibiotic)</li>

  <li>18 L 50 mg/ L ampicillin</li>

<li>Aliquot 3.5 mL into labelled round bottom falcon tubes</li>

<li>Using sterile toothpicks- pick a single colony forming unit (cfu) and inoculate tube</li>

  <li>Incubate overnight at 37°C with 215 rpm shaking</li>

<li>To 100 mL 60% glycerol stock, add 300mL overnight culture to appropriately labelled tube</li>

  <li>Store at -80°C</li>

<li>Pellet remaining cells by centrifugation in 1.5 mL tube at 13,2000 rpm for 1 min</li>

  <li>Discard supernatant and repeat</li>

<li>Resuspend pellet in Buffer P1 250 mL </li>

  <li>Add 250 L Buffer, P2 and mix by inverting 10x</li>

  <li>Add 350 L Buffer N3 and immediately mix by inverting 10x</li>

<li>Centrifuge at 13, 200 rpm for 10 minute</li>

<li>Pipet supernatant into labelled QIA prep spin column</li>

  <li>Centrifuge at 13, 200 rpm for 1 minute</li>

  <li>Discard thoroughflow </li>

<li>Wash column by adding .5 mL Buffer PB *regular for low copy number plasmids*</li>

  <li>Centrifuge at 13, 200 rpm for 1 minute</li>

  <li>Discard thoroughflow </li>

<li>Wash by adding .75 mL Buffer PE</li>

  <li>Centrifuge at 13, 200 rpm for 1 minute</li>

  <li>Discard thoroughflow </li>

  <li>Centrifuge for an addition minute to ensure removal of residual wash buffer</li>

<li>Place spin column in a clean, labelled 1.5 mL tube </li>

  <li>Add 50 LEB to center of column & let stand for 1 minute at room temperature</li>

  <li>Centrifuge at 12,000 rpm for 1 minute</li>

  <li>Store at -20℃</li>