Development of Novel Therapeutic and Diagnostic Strategies guided by Aptamer Specificity - Foundational Advance
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Aptamers
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Aptamers are short oligonucleotides, usually composed of DNA or RNA, that interact and bind to a specific target molecule with high affinity and specificity. There is functional similarly between antibodies and aptamers in the sense that they both bind specifically, but aptamers offer distinct advantages in terms of ease of production, high flexibility, and enhanced potential for application, making them very relavent in contemporary biomedical research. Aptamers form unique three-dimensional structures that facilitate specific interactions with a wide repertoire of targets, such as proteins, small molecules, and sometimes even entire cells! These interactions are due to the folding of the oligonucleotide into complex secondary and tertiary structures like hairpins, loops, and bulges, which allow aptamers to "lock" onto their targets with remarkable precision.
Aptamers are oligonucleotides, usually composed of DNA or RNA, that interact and bind to a specific target molecule with high affinity and specificity. There is functional similarly between antibodies and aptamers in the sense that they both bind specifically, but aptamers offer distinct advantages in terms of ease of production, high flexibility, and enhanced potential for application, making them very relavent in contemporary biomedical research. Aptamers form unique three-dimensional structures that facilitate specific interactions with a wide repertoire of targets, such as proteins, small molecules, and sometimes even entire cells! These interactions are due to the folding of the oligonucleotide into complex secondary and tertiary structures like hairpins, loops, and bulges, which allow aptamers to "lock" onto their targets with remarkable precision.
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<h2>What should this page contain?</h2>
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<h2>Experiments</h2>
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<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
<p>If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the <ahref="https://parts.igem.org/Main_Page">Registry</a>.</p>
<p><strong>Aim:</strong> To extract Tau plasmid from E. coli (BL21 strain) cells </p>
<p><strong>Materials Required:</strong> HiMedia Miller Luria Bertani Agar, HiMedia Miller Luria Bertani Broth, E. coli BL21 cells containg Tau plasmid, Invitrogen’s Plasmid Mini-Prep Kit, Kanamycin </p>
<p><strong>Procedure:</strong></p>
<p><em> Step 1: Streaking of bacteria containing tau plasmid on antibiotic medium </em></p>
<ul>
<li> Weigh 4g of HiMedia Miller Luria Bertani Agar and transfer into an Erlenmeyer flask </li>
<li> Measure 100ml of distilled water and pour it into the Erlenmeyer flask containing LB agar. </li>
<li> Cover the mouth of the flask with a piece of aluminium foil </li>
<li> Gently place the flask in an autoclave bag and place it in the autoclave </li>
<li> Autoclave the medium at 121°C and 15 PSI for 30-60 minutes </li>
<li> Transfer the flask into a Laminar Air Flow. The following steps are performed in a Laminar Air Flow under sterile conditions </li>
<li> Add 100µl of 100ng/ml kanamycin into 100ml of medium </li>
<li> Pour the medium into 5-7 sterilised agar plates </li>
<li> Streak a small amount of BL21 cells containing tau plasmid onto the plate using sterilised inoculation loop or pipette tip </li>
<li> Incubate the plate in an incubator set at 37°c for 12-14 hours</li>
</ul>
<p><em> Step 2: Setting up a primary culture </em></p>
<ul>
<li> Weigh 2.5g of HiMedia Miller Luria Bertani Broth and transfer into an Erlenmeyer flask </li>
<li> Measure 100ml of distilled water and pour it into the Erlenmeyer flask containing LB agar. </li>
<li> Cover the mouth of the flask with a piece of aluminium foil </li>
<li> Gently place the flask in an autoclave bag and place it in the autoclave </li>
<li> Autoclave the medium at 121°C and 15 PSI for 30-60 minutes </li>
<li> Transfer the flask into a Laminar Air Flow. The following steps are performed in a Laminar Air Flow under sterile conditions </li>
<li> Pipette out 5ml of LB broth into a 15ml centrifuge tube </li>
<li> Add 5µl of 100ng/ml kanamycin to the broth in the centrifuge tube </li>
<li> Pick a small amount of BL21 cells from the parent stock using a pipette tip or an inoculation loop and add this inoculum to the broth to set up a primary culture. For successive primary cultures the inoculum was picked up from the among colonies formed on the kanamycin-LB plate. </li>
<li> Incubate the plate in an incubator set at 37°c for 12-14 hours</li>
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<p><em>Step 3: Plasmid Extraction </em></p>
<ul>
<li> Pellet down the cells in the primary culture by centrifugation at 3,500rpm for 15-20 minutes </li>
<p> Tau plasmid was isolated from BL21 cells by following the protocol provided in Invitrogen’s Plasmid Mini-Prep Kit. The protocol has been provided here for reference. </p>
<li> Resuspend the pelleted cells in 250 µL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain. Ensure RNase A has been added to the Resuspension Solution.</li>
<li> Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA. </li>
<li> Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy. </li>
<li> Centrifuge for 5 min to pellet cell debris and chromosomal DNA. </li>
<li> Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. Close the bag with GeneJET Spin Columns tightly after each use! </li>
<li> Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. Do not add bleach to the flow-through </li>
<li> Optional: use this preliminary washing step only if EndA+ strains which have high level of nuclease activity are used. Wash the GeneJET spin column by adding 500 µL of Wash Solution I (#R1611, diluted with isopropanol) and centrifuge for 30-60 sec. Discard the flow-through. This step is essential to remove trace nuclease activity. </li>
<li> Add 500 µL of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube. </li>
<li> Repeat the wash procedure (step 8) using 500 µL of the Wash Solution. </li>
<li> Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps. </li>
<li> Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane </li>
<li> Discard the column and store the purified plasmid DNA at -20°C. </li>
