https://bridgeslab.sph.umich.edu/protocols/api.php?action=feedcontributions&feedformat=atom&user=Swat 2026-05-09T17:09:37Z User contributions MediaWiki 1.45.1 https://bridgeslab.sph.umich.edu/protocols/index.php?title=PCR_Analysis_of_Tail_DNA&diff=170 2009-06-05T18:29:47Z

<p>Swat: </p> <hr /> <div>see [[Genotyping Details]] for strain specific details<br /> <br /> ==Materials==<br /> <br /> ==Protocol==<br /> Use the following Volumes per Reaction:<br /> <br /> #Buffer: 4 uL of 5X Go-Taq buffer (&quot;Molecular Biology Stuff&quot; box in freezer) <br /> #Forward Primer: 0.4ul<br /> #Reverse Primer: 0.4ul<br /> #dNTPs: 0.4uL of 2 mM (&quot;Molecular Biology Stuff&quot; box in freezer)<br /> #Sterile water: 13.6 uL <br /> #Polymerase Go-Taq: 1 uL (6th floor in Genotype Yellow Box in freezer) <br /> #Template: 1 uL<br /> <br /> Run PCR Program (approx 2 hours).<br /> Use Cycler 1 on 6th Floor<br /> *Login: Sergey, Just press enter to Login<br /> *Under Genotype folder, pick Ingles program for Ingles genotyping<br /> *Under Genotype folder, pick regpcr program for PLT genotyping<br /> <br /> Make sure to press enter 2x once to confirm Tubes and second time to start PCR<br /> <br /> see [[Preparing an Agarose Gel]] for details on preparing a DNA gel</div>

Swat https://bridgeslab.sph.umich.edu/protocols/index.php?title=PCR_Analysis_of_Tail_DNA&diff=166 2009-06-05T16:31:54Z

<p>Swat: </p> <hr /> <div>see [[Genotyping Details]] for strain specific details<br /> <br /> ==Materials==<br /> <br /> ==Protocol==<br /> <br /> Use the following Volumes per Reaction:<br /> <br /> Buffer: 4 uL of 5X Go-Taq buffer (&quot;Molecular Biology Stuff&quot; box in freezer) <br /> <br /> Forward Primer: .4ul<br /> <br /> Reverse Primer: .4ul<br /> <br /> dNTPs: .4uL of 2 mM (&quot;Molecular Biology Stuff&quot; box in freezer)<br /> <br /> Sterile water: 13.6 uL <br /> <br /> Polymerase Go-Taq: 1 uL (6th floor in Genotype Yellow Box in freezer) <br /> <br /> Template: 1 uL<br /> <br /> <br /> <br /> Run PCR Program (approx 2 hours).<br /> Use Cycler 1 on 6th Floor<br /> <br /> Login: Sergey, Just press enter to Login<br /> <br /> Under Genotype folder, pick Ingles program for Ingles genotyping<br /> <br /> Under Genotype folder, pick regpcr program for PLT genotyping<br /> <br /> Make sure to press enter 2x once to confirm Tubes and second time to start PCR<br /> <br /> <br /> see [[Preparing an Agarose Gel]] for details on preparing a DNA gel</div>

Swat https://bridgeslab.sph.umich.edu/protocols/index.php?title=PCR_Analysis_of_Tail_DNA&diff=165 2009-06-05T16:28:20Z

<p>Swat: </p> <hr /> <div>see [[Genotyping Details]] for strain specific details<br /> <br /> ==Materials==<br /> <br /> ==Protocol==<br /> <br /> Use the following Volumes per Reaction:<br /> <br /> Buffer: 4 uL of 5X Go-Taq buffer (&quot;Molecular Biology Stuff&quot; box in freezer) <br /> <br /> Forward Primer: .4ul<br /> <br /> Reverse Primer: .4ul<br /> <br /> dNTPs: .4uL of 2 mM (&quot;Molecular Biology Stuff&quot; box in freezer)<br /> <br /> Sterile water: 13.6 uL <br /> <br /> Polymerase Go-Taq: 1 uL (6th floor in Genotype Yellow Box in freezer) <br /> <br /> Template: 1 uL<br /> <br /> <br /> see [[Preparing an Agarose Gel]] for details on preparing a DNA gel</div>

Swat https://bridgeslab.sph.umich.edu/protocols/index.php?title=PCR_Analysis_of_Tail_DNA&diff=164 2009-06-05T16:27:50Z

<p>Swat: </p> <hr /> <div>see [[Genotyping Details]] for strain specific details<br /> <br /> ==Materials==<br /> <br /> ==Protocol==<br /> <br /> Use the following volumes per reaction <br /> Buffer: 4 uL of 5X Go-Taq buffer (&quot;Molecular Biology Stuff&quot; box in freezer) <br /> Forward Primer: .4ul<br /> Reverse Primer: .4ul<br /> dNTPs: .4uL of 2 mM (&quot;Molecular Biology Stuff&quot; box in freezer) <br /> Sterile water: 13.6 uL <br /> Polymerase Go-Taq: 1 uL (6th floor in Genotype Yellow Box in freezer) <br /> Template: 1 uL<br /> <br /> <br /> see [[Preparing an Agarose Gel]] for details on preparing a DNA gel</div>

Swat https://bridgeslab.sph.umich.edu/protocols/index.php?title=DNA_Preparation_from_Tail_Clip&diff=163 2009-06-05T16:20:45Z

<p>Swat: </p> <hr /> <div>Use Qiagen DNEasy kit for Blood and Tissue Samples<br /> <br /> [edit] Protocol<br /> <br /> 1. Cut .5cm lengths of tail into a 1.5 ml microcentrifuge tube. Add 180ul Buffer ATL.<br /> <br /> 2. Add 20ul proteinase K. Mix by vortexing and incubate at 56C until tissue is completely lysed. (Overnight) <br /> <br /> 3. Vortex for 15s. Add 200ul Buffer Al to sample and vortex. Then add 200 ul ethanol (96-100%) and vortex.<br /> <br /> 4. Pipet the mixture from step 3 into DNeasy Mini spin column. Centrifuge at 8000rpm for 1 minute. Discard flow-through and collection tube. <br /> <br /> 5. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW1. Centrifuge for 1 minute at 8000rpm. Discard flow-through and collection tube. <br /> <br /> 6. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW2. Centrifuge for 3 minutes at 14000rpm. Discard flow-through and collection tube. <br /> <br /> 7. Place DNeasy Mini spin column in clean 1.5 microcentrifuge tube and pipet 200ul Buffer AE directly onto the DNeasy membrance. Incubate at room tempature for 1 minute and then centrifuge for 1 minute at 8000rpm to elute.</div>

Swat https://bridgeslab.sph.umich.edu/protocols/index.php?title=DNA_Preparation_from_Tail_Clip&diff=162 2009-06-05T16:19:14Z

<p>Swat: </p> <hr /> <div>Use Qiagen DNEasy kit for Blood and Tissue Samples<br /> <br /> [edit] Protocol<br /> <br /> 1. Cut .5cm lengths of tail into a 1.5 ml microcentrifuge tube. Add 180ul Buffer ATL.<br /> <br /> 2. Add 20ul proteinase K. Mix by vortexing and incubate at 56C until tissue is completely lysed. (Overnight) <br /> <br /> 3. Vortex for 15s. Add 200ul Buffer Al to sample and vortex. Then add 200 ul ethanol (96-100%) and vortex.<br /> <br /> 4.Pipet the mixture from step 3 into DNeasy Mini spin column. Centrifuge at 8000rpm for 1 minute. Discard flow-through and collection tube. <br /> <br /> 5. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW1. Centrifuge for 1 minute at 8000rpm. Discard flow-through and collection tube. <br /> <br /> 6. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW2. Centrifuge for 3 minutes at 14000rpm. Discard flow-through and collection tube. <br /> <br /> 7. Place DNeasy Mini spin column in clean 1.5 microcentrifuge tube and pipet 200ul Buffer AE directly onto the DNeasy membrance. Incubate at room tempature for 1 minute and then centrifuge for 1 minute at 8000rpm to elute.</div>

Swat https://bridgeslab.sph.umich.edu/protocols/index.php?title=DNA_Preparation_from_Tail_Clip&diff=161 2009-06-05T16:18:53Z

<p>Swat: </p> <hr /> <div>Use Qiagen DNEasy kit for Blood and Tissue Samples<br /> <br /> [edit] Protocol<br /> <br /> 1. Cut .5cm lengths of tail into a 1.5 ml microcentrifuge tube. Add 180ul Buffer ATL.<br /> 2. Add 20ul proteinase K. Mix by vortexing and incubate at 56C until tissue is completely lysed. (Overnight) <br /> 3. Vortex for 15s. Add 200ul Buffer Al to sample and vortex. Then add 200 ul ethanol (96-100%) and vortex.<br /> 4.Pipet the mixture from step 3 into DNeasy Mini spin column. Centrifuge at 8000rpm for 1 minute. Discard flow-through and collection tube. <br /> 5. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW1. Centrifuge for 1 minute at 8000rpm. Discard flow-through and collection tube. <br /> 6. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW2. Centrifuge for 3 minutes at 14000rpm. Discard flow-through and collection tube. <br /> 7. Place DNeasy Mini spin column in clean 1.5 microcentrifuge tube and pipet 200ul Buffer AE directly onto the DNeasy membrance. Incubate at room tempature for 1 minute and then centrifuge for 1 minute at 8000rpm to elute.</div>

Swat