Team:StanfordBrownSpelman/Lab
From 2014.igem.org
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<br />Incubate at 37°C for 1-2 hrs (<30 min for HF) | <br />Incubate at 37°C for 1-2 hrs (<30 min for HF) | ||
<br />Heat kill at 80°C for 20 minutes if proceeding to ligation | <br />Heat kill at 80°C for 20 minutes if proceeding to ligation | ||
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- | < | + | <br /><b>Verification<b> |
- | < | + | <br />Gel Casting: |
- | <br />Gel | + | <br />0.75% agarose (if DNA>1000bp) |
- | + | <br />40mL 1x TAE | |
- | + | <br />0.3 g agarose | |
- | + | <br />1 aliquot (~5μl) gel red | |
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- | + | <br />Add dry agarose to clean bottle (small enough to fit in microwave) | |
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- | 1 aliquot (~5μl) gel red | + | |
- | <br /><br />Add dry agarose to clean bottle (small enough to fit in microwave) | + | |
<br />Add 40mL 1x TAE buffer | <br />Add 40mL 1x TAE buffer | ||
<br />Microwave with cap on but loose, swish periodically, until solution is clear and smooth | <br />Microwave with cap on but loose, swish periodically, until solution is clear and smooth | ||
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<br />Pipette in gel red, directly into solution (heat stable so don’t worry about the temperature) | <br />Pipette in gel red, directly into solution (heat stable so don’t worry about the temperature) | ||
<br />Pour into gel tray, making sure that tray is oriented and tightly inserted such that leaks will not occur, and that the gel is level | <br />Pour into gel tray, making sure that tray is oriented and tightly inserted such that leaks will not occur, and that the gel is level | ||
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<br /><br />Gel Loading & Running | <br /><br />Gel Loading & Running | ||
<br />Lane 1 should be ladder; use 1kb ladder or 100bp ladder depending on the size of your DNA samples | <br />Lane 1 should be ladder; use 1kb ladder or 100bp ladder depending on the size of your DNA samples | ||
- | Digests can require more (~1.5x) than the usual amount of loading dye | + | <br />Digests can require more (~1.5x) than the usual amount of loading dye |
- | Gel Imaging (using Typhoon scanner) | + | <br /><br />Gel Imaging (using Typhoon scanner) |
- | Always scan a gel immediately after running | + | <br />Always scan a gel immediately after running |
- | Make sure the scanner area is clean; wipe ONLY with 70% ethanol (or DI) and kimtech wipes | + | <br />Make sure the scanner area is clean; wipe ONLY with 70% ethanol (or DI) and kimtech wipes |
- | Gel should be placed on scanner face-up. That is, the wells should be oriented up, the same way the gel is oriented in the gel box | + | <br />Gel should be placed on scanner face-up. That is, the wells should be oriented up, the same way the gel is oriented in the gel box |
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<br /><br />Gel Extraction & Cleanup | <br /><br />Gel Extraction & Cleanup | ||
<br />Make sure to place gel on transilluminator face down (wells toward the glass) | <br />Make sure to place gel on transilluminator face down (wells toward the glass) | ||
- | Remove as much excess gel matrix as possible without overexposing DNA to UV | + | <br />Remove as much excess gel matrix as possible without overexposing DNA to UV |
<br /><br />For cleanup, follow protocol for using the Wizard PCR Cleanup Kit, found below in PCR section | <br /><br />For cleanup, follow protocol for using the Wizard PCR Cleanup Kit, found below in PCR section | ||
- | <br /><br />Ligation (adapted from openwetware ligation protocol): | + | <br /><br /><b>Ligation</b> (adapted from openwetware ligation protocol): |
- | 10 μl Recipe | + | <br />10 μl Recipe: |
- | 30-50 ng vector DNA ( | + | <br />30-50 ng vector DNA (<a href="http://www.insilico.uni-duesseldorf.de/Lig_Input.html">A calculator to make life easy</a>) |
- | < | + | <br />1μl (10%) 10X T4 DNA ligase buffer |
- | + | <br />0.5μl (.5%) T4 DNA ligase | |
- | A calculator to make life easy | + | <br />Top up w/ qH20 up to 10uL |
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- | + | <br /><br />Procedure | |
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- | 1μl (10%) 10X T4 DNA ligase buffer | + | |
- | 0.5μl (.5%) T4 DNA ligase | + | |
- | Top up w/ qH20 up to 10uL | + | |
- | <br />Procedure | + | |
<br />Usually heat inactivation of digests is sufficient; difficult ligations might require a proper cleanup | <br />Usually heat inactivation of digests is sufficient; difficult ligations might require a proper cleanup | ||
- | As often as possible, use isolated inserts and vectors to avoid unwanted ligations | + | <br />As often as possible, use isolated inserts and vectors to avoid unwanted ligations |
- | If the reaction needs to be greater than 10μl, adjust amount of 10X ligase buffer and T4 DNA ligase so that they remain at 1% and .5% by volume, respectively | + | <br />If the reaction needs to be greater than 10μl, adjust amount of 10X ligase buffer and T4 DNA ligase so that they remain at 1% and .5% by volume, respectively |
- | For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. | + | <br />For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. |
- | For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours(alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). | + | <br />For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). |
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- | Chemically Competent Transformation | + | <b>Chemically Competent Transformation</b> |
<br />Materials | <br />Materials | ||
<br />1 aliquot of competent cells | <br />1 aliquot of competent cells |
Revision as of 01:09, 16 October 2014