Team:Glendale CC AZ/Protocols/RestrictionDigest
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+ | <body> | ||
+ | <div class="content-area"> <article> | ||
+ | </article> | ||
+ | <img style="border: 0px solid ; width: 50px; height: 40px;" alt="iGEM" src="http://s21.postimg.org/ff5nkjy9v/IGEM_basic_Logo_stylized.png" align="left"> | ||
+ | <h4>Glendale Community College Arizona<img | ||
+ | style="width: 200px; height: 58px;" alt="GCC" | ||
+ | src="https://static.igem.org/mediawiki/2013/f/f1/Gcclogo.gif" align="right"> | ||
+ | </h4> | ||
+ | |||
+ | |||
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+ | |||
+ | |||
+ | <h2><p>Protocols</p></h2><img style="border: 0px solid ; width: 400px; height: 200px;" alt="iGEM" src="https://static.igem.org/mediawiki/igem.org/1/19/Tubes2GCC.JPG" align="right"> | ||
+ | |||
+ | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/GrowthCurve">Growth Curve Assay</a></p> | ||
+ | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/NaCl">NaCl Growth Curve Assay</a></p> | ||
+ | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/SurivalGrowth">Survival Growth Assay</a></p> | ||
+ | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/AlkalineLysis">Alkaline Lysis Plasmid Miniprep </a></p> | ||
+ | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/RestrictionDigest">Restriction Digest</a></p> | ||
+ | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/DNAIsolation">DNA Isolation</a></p> | ||
+ | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/Bioinformatics">Bioinformatics</a></p> | ||
+ | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/Ligation">Ligation</a></p> | ||
+ | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/Transformation">Transformation</a></p> | ||
+ | |||
+ | |||
+ | <!--/.content-area--> | ||
+ | </body> | ||
+ | </html> | ||
+ | |||
+ | ==== Restriction Digest Protocol ==== | ||
+ | |||
+ | Protocol can be found at: http://parts.igem.org/Help:Protocols/Restriction_Digest | ||
+ | |||
+ | ==== Materials ==== | ||
-Ice and bucket/container | -Ice and bucket/container | ||
- | - | + | -Part 1 DNA |
- | + | -Part 2 DNA | |
- | -Part | + | -Linearized plasmid backbone (25ng/µl) |
- | -Linearized plasmid backbone (25ng/ | + | |
-dH2O | -dH2O | ||
- | -NEB Buffer 2, 3.1,or Ecor1 | + | -NEB Buffer 2, 3.1,or Ecor1 |
-BSA (Not needed with 3.1 buffer) | -BSA (Not needed with 3.1 buffer) | ||
- | -Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, | + | -Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, DnpI |
- | -Thermal cycler | + | -Thermal cycler |
+ | |||
+ | ==== Procedure ==== | ||
+ | |||
+ | 1. Keep all enzymes and buffers used on ice. | ||
+ | |||
+ | 2. Thaw NEB Buffer and BSA (if needed- NEB buffers with .1 do not require additional BSA ie. NEB buffer 3.1) in room temperature water. | ||
+ | |||
+ | 3. Add 250ng of DNA to the appropriately labeled tube. | ||
+ | |||
+ | 4. In the Part 1 tube: Add 0.5µL of EcoRI, and 0.5µL of SpeI. | ||
+ | |||
+ | 5. In the Part 2 tube: Add 0.5µL of XbaI, and 0.5µL of PstI. | ||
+ | |||
+ | 6. In the plasmid tube: Add 0.5µL of EcoRI, 0.5µL of PstI, and 0.5µL of Dpn1. | ||
+ | |||
+ | 7. Add 0.5µL of BSA to each tube. (If required) | ||
+ | 8. Add dH20 to a final volume of 20µL. | ||
- | + | 9. Preset thermal cycler program to run the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes. |
Latest revision as of 02:35, 28 September 2013
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Glendale Community College Arizona
Protocols
Restriction Digest Protocol
Protocol can be found at: http://parts.igem.org/Help:Protocols/Restriction_Digest
Materials
-Ice and bucket/container -Part 1 DNA -Part 2 DNA -Linearized plasmid backbone (25ng/µl) -dH2O -NEB Buffer 2, 3.1,or Ecor1 -BSA (Not needed with 3.1 buffer) -Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, DnpI -Thermal cycler
Procedure
1. Keep all enzymes and buffers used on ice.
2. Thaw NEB Buffer and BSA (if needed- NEB buffers with .1 do not require additional BSA ie. NEB buffer 3.1) in room temperature water.
3. Add 250ng of DNA to the appropriately labeled tube.
4. In the Part 1 tube: Add 0.5µL of EcoRI, and 0.5µL of SpeI.
5. In the Part 2 tube: Add 0.5µL of XbaI, and 0.5µL of PstI.
6. In the plasmid tube: Add 0.5µL of EcoRI, 0.5µL of PstI, and 0.5µL of Dpn1.
7. Add 0.5µL of BSA to each tube. (If required)
8. Add dH20 to a final volume of 20µL.
9. Preset thermal cycler program to run the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.