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Team:Pitt/Skin Probiotic/Cathelicidin/Methods
From 2014.igem.org
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| - | <li> | + | <li> DNA Digest with <a href="http://www.thermoscientificbio.com/restriction-and-modifying-enzymes/restriction-enzymes/reaction-conditions-for-fastdigest-enzymes-chart/">Thermo Scientific FastDigest Restriction Enzymes</a><br> |
1.1. Digest Insert with XbaI and PstI or EcoRI and SpeI<br> | 1.1. Digest Insert with XbaI and PstI or EcoRI and SpeI<br> | ||
1.2. Digest Vector Backbone with SpeI and PstI or EcoRI and XbaI</li> | 1.2. Digest Vector Backbone with SpeI and PstI or EcoRI and XbaI</li> | ||
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3.2. Perform Gel Purification using <a href="https://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/101/wizard%20sv%20gel%20and%20pcr%20clean-up%20system%20protocol.pdf">Promega SV Wizard Gel and PCR Clean Up System</a><br> | 3.2. Perform Gel Purification using <a href="https://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/101/wizard%20sv%20gel%20and%20pcr%20clean-up%20system%20protocol.pdf">Promega SV Wizard Gel and PCR Clean Up System</a><br> | ||
3.3. Measure DNA concentration using <a href="http://www.fishersci.com/ecomm/servlet/fsproductdetail_10652_9988497__-1_0">Fisher Scientific Nanodrop 2000</a></li> | 3.3. Measure DNA concentration using <a href="http://www.fishersci.com/ecomm/servlet/fsproductdetail_10652_9988497__-1_0">Fisher Scientific Nanodrop 2000</a></li> | ||
| - | <li> | + | <li> Ligation of Vector Backbone and Insert with <a href="https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202">T4 DNA Ligase</a></li> |
| - | <li> | + | <li> Transform Ligation Products into <a href="http://tools.lifetechnologies.com/content/sfs/manuals/oneshot_mach1_man.pdf">Mach1 Competent Cells</a></li> |
<li> Spread Transformation Mix onto selection plate<br> | <li> Spread Transformation Mix onto selection plate<br> | ||
6.1. Selected on either Ampicillin or Chloramphenicol depending on resistance gene<br> | 6.1. Selected on either Ampicillin or Chloramphenicol depending on resistance gene<br> | ||
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<li> Select colony from plate and create 5 mL liquid cultures<br> | <li> Select colony from plate and create 5 mL liquid cultures<br> | ||
7.1. Incubate 5-8 hours</li> | 7.1. Incubate 5-8 hours</li> | ||
| - | <li> Extract Plasmids using Qiagen Miniprep Kit<br> | + | <li> Extract Plasmids using <a href="http://www.qiagen.com/resources/resourcedetail?id=331740ca-077f-4ddd-9e5a-2083f98eebd5&lang=en">Qiagen Miniprep Kit</a><br> |
8.1. Measure DNA concentration using Fisher Scientific Nanodrop 2000</li> | 8.1. Measure DNA concentration using Fisher Scientific Nanodrop 2000</li> | ||
<li> Diagnostic DNA Digest<br> | <li> Diagnostic DNA Digest<br> | ||
Revision as of 20:02, 17 October 2014
Methods for Cathelicidin
- DNA Digest with Thermo Scientific FastDigest Restriction Enzymes
1.1. Digest Insert with XbaI and PstI or EcoRI and SpeI
1.2. Digest Vector Backbone with SpeI and PstI or EcoRI and XbaI - Gel Electrophoresis
2.1. Create 1% agarose gel with EtBr
2.2. Load gel lanes with Invitrogen 1kb Plus ladder and restriction digest products
2.3. Run Gel until band separation is visible - Gel Extraction and Purification
3.1. Image the gel and extract the desired bands
3.2. Perform Gel Purification using Promega SV Wizard Gel and PCR Clean Up System
3.3. Measure DNA concentration using Fisher Scientific Nanodrop 2000 - Ligation of Vector Backbone and Insert with T4 DNA Ligase
- Transform Ligation Products into Mach1 Competent Cells
- Spread Transformation Mix onto selection plate
6.1. Selected on either Ampicillin or Chloramphenicol depending on resistance gene
6.2. Incubate overnight
- Select colony from plate and create 5 mL liquid cultures
7.1. Incubate 5-8 hours - Extract Plasmids using Qiagen Miniprep Kit
8.1. Measure DNA concentration using Fisher Scientific Nanodrop 2000 - Diagnostic DNA Digest
9.1. Digest Plasmids with XbaI and PstI
9.2. Run Digestion products on 1% agarose gel with EtBr
9.3. Check for correct band lengths
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