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| <h2>Experiments & Protocols</h2> | | <h2>Experiments & Protocols</h2> |
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− | <h4>1 - Bacteria Media w/ agar (solid media)</h4> | + | <h3>Bacteria Media w/ agar (solid media)</h3> |
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| <h5>Materials</h5> | | <h5>Materials</h5> |
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| <li>Bacto Peptone</li> | | <li>Bacto Peptone</li> |
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| + | <h5>Procedure</h5> |
| <ol type=”1”> | | <ol type=”1”> |
| <li>Add 250mL of deionized water to the 500mL bottle. </li> | | <li>Add 250mL of deionized water to the 500mL bottle. </li> |
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| <li>Let dishes sit overnight.</li> | | <li>Let dishes sit overnight.</li> |
| </ol> | | </ol> |
| + | � |
| + | <h3>2 - Dynabead Prep</h3> |
| | | |
| + | <h5>Materials</h5> |
| + | <li>B&W wash Buffer (2X and 1X)</li> |
| + | <li>Dynabeads</li> |
| + | <li>DNA solution</li> |
| + | <li>Magnets</li> |
| + | <li>PCR Tubes</li> |
| + | |
| + | <h5>Procedure</h5> |
| + | <ol type=”1”> |
| + | <li>Pipette 50 microL of dynabeads in PCR tubes (3 of them)</li> |
| + | <li>Wash beads by pipetting in 200 microL of 1X B&W buffer</li> |
| + | <li>Vortex PCR tubes for about 30 seconds</li> |
| + | <li>Place a magnet at the bottom of the tube until a solid pellet of dynabeads forms at the bottom</li> |
| + | <li>Pipette out the supernatant leaving the pellet intact at the bottom</li> |
| + | <li>Repeat steps 2-5 two times (performed a total 3 times)</li> |
| + | <li>To each tube add 100 microL 2X buffer, 5 microL DNA (100 microM), and 95 microL of water</li> |
| + | <li>Incubate on stirring apparatus/agitator at room temperature for 15 minutes</li> |
| + | <li>Wash the beads again by repeating steps 2-5 three times</li> |
| + | <li>Add 105 microL 1X buffer to the tubes to resuspend the beads</li> |
| + | <li>Freeze PCR tubes at -20 degrees Celsius until they are needed </li> |
| + | </ol> |
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| + | <h3>3 - Bacterial Transformation</h3> |
| + | |
| + | <li>For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.</li> |
| + | <li>For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.</li> |
| + | <li>Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.</li> |
| + | <li>Place the mixture on ice for 30 minutes. Do not mix.</li> |
| + | <li>Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.</li> |
| + | <li>Place on ice for 5 minutes. Do not mix.</li> |
| + | <li>Pipette 950 µl of room temperature SOC into the mixture.</li> |
| + | <li>Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.</li> |
| + | <li>Warm selection plates to 37°C.</li> |
| + | <li>Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.</li> |
| + | <li>Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. </li> |
| + | <li>Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.</li> |
| + | |
| + | <a href=”https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987”>Protocol Retrieved From</a> |
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| + | � |
| + | <h3>4 - Colony PCR of pSB1C3 with TdT Mutant Inserts</h3> |
| + | |
| + | <h5>Materials</h5> |
| + | <li>VR Primer</li> |
| + | <li>VF2 Primer</li> |
| + | <li>pSB1C3 colonies</li> |
| + | <li>ddH20</li> |
| + | <li>10 mm dNTPs</li> |
| + | <li>Taq Polymerase</li> |
| + | <li>10X Standard Taq Polymerase Buffer</li> |
| + | <li>6X Loading Dye</li> |
| + | <li>DNA Ladder</li> |
| + | <li>1% Agarose Gel</li> |
| + | |
| + | <h5>Procedure</h5> |
| + | <ol type=”1”> |
| + | <li>Use a pipette tip to scrape off a colony and place the colony in 200 microL of ddH20</li> |
| + | <li>Take 21 microL of bacteria water and add it to a PCR tube</li> |
| + | <li>Add half a microliter of primer VR and another half microliter of primer VF2 to PCR tube</li> |
| + | <li>Add half a microliter of 10 mm dNTPs, 0.125 microliters of Taq Polymerase, and the appropriate amount of Standard Taq Polymerase Buffer (Usually 2.5 for a 25 microliter reaction)</li> |
| + | <li>Run in PCR machine with desired thermal cycling</li> |
| + | <li>Prepare a 1% agarose gel if one is not available</li> |
| + | <li>Prepare sample for gel electrophoresis using a proper proportion of 6X loading dye to sample </li> |
| + | <li>Load DNA ladder and sample into gel and run gel</li> |
| + | <li>Analyze gel using U.V. light and determine the approximate length of the amplified sequence</li> |
| + | </ol> |
| + | |
| + | <a href=”https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273”>Procedure adapted from here</a> |
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| + | <h3>5 - Dynabead Cleanamp</h3> |
| + | |
| + | <h5>Materials</h5> |
| + | <li>10x TdT buffer</li> |
| + | <li>2.5 mM CoCl2</li> |
| + | <li>Oligo</li> |
| + | <li>50 mM CleanAmp dATP</li> |
| + | <li>100 mM dATP</li> |
| + | <li>TdT</li> |
| + | <li>dH2O</li> |
| + | |
| + | |
| + | <h5>Procedure</h5> |
| + | <ol type=”1”> |
| + | <li>Preheat water baths to 37 celsius and 95 celsius. |
| + | <li>Mix: |
| + | - 5 µl 10x TdT buffer, |
| + | - 5 µl 2.5 mM CoCl2, |
| + | - 1.3 µl of 43 mer oligo ( 1 µg at 813 ng/µl), |
| + | - 0.4 µl 50 mM CleanAmp dATP, |
| + | - 1 µl TdT ( 20 units at 20000 units/mL), |
| + | - dH2O to final volume of 50 mL. |
| + | <li>Split into two separate equal volume aliquots. |
| + | <li>Incubate at 37 celsius for 30 minutes. |
| + | <li>Incubate 1 aliquot at 95 celsius for 15 minutes. |
| + | <li>For the non-CleanAmp dATP, replace CleanAmp dATP with 0.2 µl 100 mM dATP. |
| + | <li>For the negative control, remove the volume of TdT. |
| + | <li>Store at -20 celsius. |
| + | </ol> |
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| + | <h3>6 - Double Digest of PET, PSB, TdT variants</h3> |
| + | |
| + | <h5>Materials</h5> |
| + | <li>PET28</li> |
| + | <li>PSB1C3</li> |
| + | <li>Nuclease-free water</li> |
| + | <li>EcoR1-HF</li> |
| + | <li>Pst</li> |
| + | <li>XbaI</li> |
| + | <li>BamhI</li> |
| + | <li>TdT (TdT del1-27, del26-143, & GIP subAAA 213-215)</li> |
| + | <li>NEBuffer 3.1</li> |
| + | <li>NEBuffer 2.1</li> |
| + | |
| + | <h5>Procedure</h5> |
| + | <ol type=”1”> |
| + | <li>Digest of PET28 and PSB1C3</li> |
| + | <ol type=”i”> |
| + | <li>Mix together these materials in this order for PET28 tube:</li> |
| + | <ol type=”a”> |
| + | <li>3.1 buffer---5uL</li> |
| + | <li>Water---32.2uL</li> |
| + | <li>XbaI---1uL</li> |
| + | <li>BamhI---1uL</li> |
| + | <li>PET28---10.8uL </li> |
| + | </ol> |
| + | <li>Mix together these materials in this order for PSB1C3 tube:</li> |
| + | <ol type=”a”> |
| + | <li>2.1 buffer---5uL</li> |
| + | <li>Water---31.2uL </li> |
| + | <li>EcoRI---1uL</li> |
| + | <li>PstI---1uL</li> |
| + | <li>PSB1C3---11.8uL</li> |
| + | </ol> |
| + | <li>Incubate both tubes at 37˚C for 1 hour</li> |
| + | </ol> |
| + | <li>Digestion of TdT sequences</li> |
| + | <ol type=”i”> |
| + | <li>Take three tubes and mix together these ingredients for each:</li> |
| + | <ol type=”a”> |
| + | <li>3.1 buffer---1.5uL</li> |
| + | <li>Water---2.5uL</li> |
| + | <li>XbaI---0.5uL</li> |
| + | <li>BamhI---0.5uL</li> |
| + | </ol> |
| + | <li>Place each TdT variant in each tube </li> |
| + | <li>Take three more tubes and mix together these ingredients for each:</li> |
| + | <ol type=”a”> |
| + | <li>2.1 buffer---1.5uL</li> |
| + | <li>Water---2.5uL</li> |
| + | <li>EcoRI---0.5uL</li> |
| + | </li>PstI---0.5uL</li> |
| + | </ol> |
| + | |
| + | <li>Place each TdT variant in each tube</li> |
| + | <li>Incubate both tubes at 37˚C for 1 hour</li> |
| + | <li>Purify TdT DNA</li> |
| + | <ol type=”a”> |
| + | <li>tubes containing EcoRI and PstI are purified by incubating at 80˚C for 20 minutes</li> |
| + | <li>tubes containing XbaI and PstI are purified using life technologies "Procedure for Purifying PCR Products" (in orange folder)</li> |
| + | </ol> |
| + | </ol> |
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| + | <h3>7 - Immobilizing Disulfide Modified Oligos to Mercaptosilane Treated Glass Slides</h3> |
| + | |
| + | <h5>Materials</h5> |
| + | <li>Untreated Glass Slides or Glass Coverslips</li> |
| + | <li>Disulfide Modified Oligonucleotides (Ordered from IDT with the C6 S-S 5’ Thiol Modifier)</li> |
| + | <li>500 mM Sodium Bicarbonate Buffer (pH 9.0)</li> |
| + | <li>Anhydrous Ethanol</li> |
| + | <li>25% Ammonium Hydroxide</li> |
| + | <li>1% 3-mercaptopropyl trimethoxysilane, 95% Ethanol, 16 mM acetic acid (pH 4.5)</li> |
| + | <li>95% Ethanol, 16 mM acetic acid (pH 4.5)</li> |
| + | <li>10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20</li> |
| + | <li>Running RO water or ddH20</li> |
| + | <li>Vacuum Oven</li> |
| + | <li>Vacuum Pump</li> |
| + | <li>Fume Hood</li> |
| + | <li>Petri dishes</li> |
| + | <li>Parafilm</li> |
| + | |
| + | <ol type=”1”> |
| + | <li>Place untreated glass slides in a clean Petri dish</li> |
| + | <li>Submerge glass slides in 25% Ammonium Hydroxide overnight</li> |
| + | <li>Rinse the same glass slides under running RO water for 10 minutes</li> |
| + | <li>Rinse the slides with Anhydrous Ethanol </li> |
| + | <li>Immerse the slides in 1% 3-mercaptopropyl trimethoxysilane, 95% Ethanol, 16 mM acetic acid (pH 4.5) for 30 - 45 minutes</li> |
| + | <li>Rinse the same slides with 95% Ethanol, 16 mM acetic acid (pH 4.5)</li> |
| + | <li>Cure the glass slides in a vacuum oven at 150C for 2 hours</li> |
| + | <li>While slides are curing, suspend 1 - 40 uM of disulfide modified DNA in 150 uL of of 500 mM Sodium Bicarbonate Buffer (pH 9.0)</li> |
| + | <li>Remove cured slides from vacuum oven, and array disulfide modified DNA suspended in 500 mM Sodium Bicarbonate Buffer (pH 9.0) onto slides. Various arrays, ranging from 5 x 5 lattices using 2 uL dots to 1 large 50 uL dot were used </li> |
| + | <li>Place the slides in a moist environment, such as on a rack in a 30C water bath, overnight</li> |
| + | <li>Clean the slides using three washes of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20</li> |
| + | <li>Store the slides in 4C temperature</li> |
| + | </ol> |
| + | |
| + | |
| + | <a href=”http://xobi.net/Lib/AnalBio99v266p23.pdf”>Protocol Retrieved from Here</a> |
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| + | <h3>8 - RNA Ligase Reaction</h3> |
| + | |
| + | <h5>Materials</h5> |
| + | <li>1 ug Single Stranded DNA with 5’ Phosphate (X mer) [Retrieved as an IDT Custom Oligo]</li> |
| + | <li>1 ug Single Stranded DNA (Y mer) [Retrieved as an IDT Custom Oligo or can be any primer]</li> |
| + | <li>T4 RNA Ligase 1</li> |
| + | <li>T4 RNA Ligase 1 Reaction Buffer</li> |
| + | <li>PEG 8000</li> |
| + | <li>ATP</li> |
| + | <li>10 mM Tris-HCl pH 8.0, 2.5 mM EDTA</li> |
| + | |
| + | <h5>Procedure</h5> |
| + | <ol type=”1”> |
| + | <li>Make a 20 uL reaction that contains the following:</li> |
| + | <ul> |
| + | <li>1 ug of ssDNA with 5’ Phosphate</li> |
| + | <li>1 ug of ssDNA (Any primer or oligo)</li> |
| + | <li>25% PEG 8000</li> |
| + | <li>1 mM ATP</li> |
| + | <li>1X T4 RNA Ligase 1 Reaction Buffer</li> |
| + | <li> uL of T4 RNA Ligase 1</li> |
| + | </ul> |
| + | <li>Incubate the mixture overnight for 16-18 hours at room temperature</li> |
| + | <li>Terminate the reaction by adding 40 uL of 10 mM Tris-HCl pH 8.0, 2.5 mM EDTA</li> |
| + | </ol> |
| + | |
| + | <a href=”https://www.neb.com/protocols/1/01/01/ligation-of-an-oligo-to-single-stranded-dna-cdna-using-t4-rna-ligase-1”>Procedure Retrieved from Here”</a> |
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| + | � |
| + | <h3>9 - Gel Extraction</h3> |
| + | |
| + | <h5>Materials</h5> |
| + | <li>DNA sample ran through agarose gel</li> |
| + | <li>Sharp edge, like scalpel or gel cutter</li> |
| + | <li>Qiagen Buffers QG, PE, EB</li> |
| + | <li>Isopropanol</li> |
| + | |
| + | <h5>Procedure</h5> |
| + | <ol type=”1”> |
| + | <li>Prewarm waterbath to 50°C</li> |
| + | <li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</li> |
| + | <li>Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl)</li> |
| + | <li>Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.</li> |
| + | <li>After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).</li> |
| + | <li>Add 1 gel volume of isopropanol to the sample and mix.</li> |
| + | <li>Place a QIAquick spin column in a provided 2 ml collection tube.</li> |
| + | <li>To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.</li> |
| + | <li>Discard flow-through and place QIAquick column back in the same collection tube. |
| + | (Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.</li> |
| + | <li>To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. |
| + | Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm).</li> |
| + | <li>Place QIAquick column into a clean 1.5 ml microcentrifuge tube.</li> |
| + | <li>To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed.</li> |
| + | </ol> |
| + | |
| + | <a href=”http://www.aun.edu.eg/molecular_biology/gene&protein/Gel%20extraction-Qiagen.pdf”>Procedure Retrieved from Here”</a> |
| + | |
| + | � |
| + | <h3>10 - Miniprep</h3> |
| + | |
| + | <h5>Materials</h5> |
| + | <li>5mL of overnight bacteria culture</li> |
| + | <li>Qiagen Buffers P1, P2, N3, PE, EB</li> |
| + | |
| + | <h5>Procedure</h5> |
| + | <ol type=”1”> |
| + | <li>Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.</li> |
| + | <li>Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix</li> |
| + | <li>Add 350 µl Buffer N3 and invert the tube immediately but gently 4–6 times.</li> |
| + | <li>Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.</li> |
| + | <li>Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.</li> |
| + | <li>Centrifuge for 30–60 s. Discard the flow-through. |
| + | (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.</li> |
| + | <li>Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.</li> |
| + | <li>Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.</li> |
| + | <li>Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl </li> |
| + | <li>Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.</li> |
| + | </ol> |
| + | |
| + | |
| + | <a href=”http://stanxterm.aecom.yu.edu/wiki/data/Product_manuals_attach/qiagenmini.pdf”>Procedure Retrieved from Here”</a> |
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| + | <h3>11 - PCR Purification</h3> |
| + | |
| + | <h5>Materials</h5> |
| + | <li>PCR Reaction to be purified</li> |
| + | <li>Qiagen Buffers PB, PE, EB</li> |
| + | |
| + | <h5>Procedure</h5> |
| + | <ol type=”1”> |
| + | <li>Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.</li> |
| + | <li>If pH indicator has been added to Buffer PB, check that the color of the mixture is yellow.</li> |
| + | <li>Place a QIAquick spin column in a provided 2 ml collection tube.</li> |
| + | <li>To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.</li> |
| + | <li>Discard flow-through. Place the QIAquick column back into the same tube.</li> |
| + | <li>To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.</li> |
| + | <li>Discard flow-through and place the QIAquick column back in the same tube.</li> |
| + | <li>Centrifuge the column for an additional 1 min.</li> |
| + | <li>Place QIAquick column in a clean 1.5 ml microcentrifuge tube.</li> |
| + | <li>To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min.</li> |
| + | </ol> |
| + | |
| + | |
| + | <a href=”https://static.igem.org/mediawiki/2012/a/a3/QIAquick_PCR-purification.pdf”>Procedure Retrieved from Here”</a> |
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| + | <h3>12 - TdT Nucleotide Addition</h3> |
| + | |
| + | <h5>Materials</h5> |
| + | <li>DNA source - commercially made oligo's</li> |
| + | <li><a href=”https://www.neb.com/products/m0315-terminal-transferase”> Terminal Transferase</a></li> |
| + | <li><a href=”http://www.trilinkbiotech.com/cart/scripts/prodView.asp?idproduct=4665”>CleanAmp dNTP's</a></li> |
| + | |
| + | <h5>Procedure</h5> |
| + | <li>Repeat for each nucleotide</li> |
| + | <ol type=”1”> |
| + | <li>Pre-heat water baths to 37°C and 95°C</li> |
| + | <li>Mix:</li> |
| + | </ol> |
| + | <ol type=”i”> |
| + | <li>5.0 μL 10X TdT Buffer</li> |
| + | <li>5.0 μL 2.5 mM CoCl2 solution</li> |
| + | <li>1μg 43 mer oligo (1.3μL of 813 ng/μL)</li> |
| + | <li>0.4 μL 50mM CleanAmp dATP</li> |
| + | <li>20 Units TdT (1 μL of 20,000 U/μL)</li> |
| + | <li>dH2O to final volume of 50 μL</li> |
| + | <li>Incubate at 37°C for 30 minutes.</li> |
| + | <li>Prepare 2 equal volume aliquots of the mixture (25 μL each).</li> |
| + | <li>Incubate 1 aliquot at 95°C for 15 minutes.</li> |
| + | <li>Add additional 10 U TdT (0.5 μL of 20,000 U/μL) to both aliqouts.</li> |
| + | <li>Cool 95°C water bath to 70°C.</li> |
| + | <li>Incubate at 37°C for 60 minutes.</li> |
| + | <li>Heat inactivate at 70°C for 10 minutes.</li> |
| + | <li>Store at -20°C.</li> |
| + | </ol> |
| + | |
| + | <li>Controls</li> |
| + | <ol type=”1”> |
| + | <li>Negative (No TdT)</li> |
| + | <ol type=”i”> |
| + | <li>Pre-heat water baths to 37°C and 70°C</li> |
| + | <li>Mix:</li> |
| + | </ol> |
| + | <ol type=”a”> |
| + | <li>5.0 μL 10X TdT Buffer</li> |
| + | <li>5.0 μL 2.5 mM CoCl2 solution</li> |
| + | <li>500 ng 24-mer oligo (0.9 μL of 568.8 ng/μL)</li> |
| + | <li>1.0 μL 10mM dATP</li> |
| + | <li>dH2O to final volume of 50 μL</li> |
| + | <li>Incubate at 37°C for 60 minutes</li> |
| + | <li>Heat inactivate at 70°C for 10 minutes.</li> |
| + | <li>Store at -20°C.</li> |
| + | </ol> |
| + | |
| + | </li>Five Minute Incubation Time</li> |
| + | <ol type=”i”> |
| + | <li>Pre-heat water baths to 37°C and 70°C</li> |
| + | <li>Mix:</li> |
| + | </ol> |
| + | <ol type=”a”> |
| + | <li>5.0 μL 10X TdT Buffer</li> |
| + | <li>5.0 μL 2.5 mM CoCl2 solution</li> |
| + | <li>500 ng 24-mer oligo (0.9 μL of 568.8 ng/μL)</li> |
| + | <li>1.0 μL 10mM dATP</li> |
| + | <li>10 Units TdT (0.5 μL of 20,000 U/μL)</li> |
| + | <li>dH2O to final volume of 50 μL</li> |
| + | <li>Incubate at 37°C for 5 minutes</li> |
| + | <li>Heat inactivate at 70°C for 10 minutes.</li> |
| + | <li>Store at -20°C.</li> |
| + | </ol> |
| + | </li>Sixty Minute Incubation Time</li> |
| + | <ol type=”i”> |
| + | <li>Pre-heat water baths to 37°C and 70°C</li> |
| + | <li>Mix:</li> |
| + | </ol> |
| + | <ol type=”a”> |
| + | <li>5.0 μL 10X TdT Buffer</li> |
| + | <li>5.0 μL 2.5 mM CoCl2 solution</li> |
| + | <li>500 ng 24-mer oligo (0.9 μL of 568.8 ng/μL)</li> |
| + | <li>1.0 μL 10mM dATP</li> |
| + | <li>10 Units TdT (0.5 μL of 20,000 U/μL)</li> |
| + | <li>dH2O to final volume of 50 μL</li> |
| + | <li>Incubate at 37°C for 60 minutes</li> |
| + | <li>Heat inactivate at 70°C for 10 minutes.</li> |
| + | <li>Store at -20°C.</li> |
| + | |
| + | |
| + | |
| + | |
| + | <h3>Non-Radioactive phosphorlyation with T4 PNK</h3> |
| + | |
| + | <h5>Procedure</h5> |
| + | <ol type=”1”> |
| + | <li>Mix the following:</li> |
| + | </ol> |
| + | <ul> |
| + | <li>up to 300 pmol of single stranded oligo (43mer)</li> |
| + | <li>5 microliter of T410X PNK Buffer</li> |
| + | <li>5 microliter of 10 mM ATP</li> |
| + | <li>1 microliter of PNK</li> |
| + | <li>ddH20 upto a total volume of 50 uL</li> |
| + | </ul> |
| + | |
| + | <a href=”https://www.neb.com/protocols/1/01/01/non-radioactive-phosphorylation-with-t4-pnk-or-pnk3-phosphatase-minus”>Procedure Retrieved from Here</a> |
| + | |
| <h5>What should this page contain?</h5> | | <h5>What should this page contain?</h5> |
| <ul> | | <ul> |