Team:UC Davis/Protocols
From 2011.igem.org
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+ | <html> | ||
+ | <div class="floatbox"> | ||
+ | <h1>Restriction Enzyme Double Digest</h1> | ||
- | + | Restriction Enzyme Double Digest | |
- | + | Materials | |
* 22 uL dH2O | * 22 uL dH2O | ||
* 1 uL BSA | * 1 uL BSA | ||
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* 1 uL Enzyme 2 | * 1 uL Enzyme 2 | ||
- | + | Buffer Compatibility Chart | |
{| border="1" | {| border="1" | ||
! | ! | ||
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* Run on a gel and extract product. | * Run on a gel and extract product. | ||
- | + | </div> | |
- | = | + | <div class="floatbox"> |
+ | <h1>Gel Extraction/Purification Procedure</h1> | ||
+ | |||
+ | Materials | ||
* GeneJET Gel Extraction Kit | * GeneJET Gel Extraction Kit | ||
* Binding Buffer (1 uL for every mg of agarose gel) | * Binding Buffer (1 uL for every mg of agarose gel) | ||
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* 50 uL of Elution Buffer | * 50 uL of Elution Buffer | ||
- | + | Procedure | |
* Add the binding buffer to the gel slice in a microcentrifuge tube. | * Add the binding buffer to the gel slice in a microcentrifuge tube. | ||
* Incubate the gel mixture at 50-60 °C for 10 minutes (until melted). | * Incubate the gel mixture at 50-60 °C for 10 minutes (until melted). | ||
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* Centrifuge for 1 minute and collect the flow-through. | * Centrifuge for 1 minute and collect the flow-through. | ||
- | + | </div> | |
- | = | + | <div class="floatbox"> |
+ | <h1>Transformations</h1> | ||
+ | |||
+ | Materials | ||
* Competent cells | * Competent cells | ||
* DNA template | * DNA template | ||
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* LB+antibiotic plates | * LB+antibiotic plates | ||
- | + | Procedure | |
* Thaw competent cells on ice. | * Thaw competent cells on ice. | ||
* Transfer 50 uL of competent cells to chilled falcon tubes. | * Transfer 50 uL of competent cells to chilled falcon tubes. | ||
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* Incubate at 37 °C for 1 hour. | * Incubate at 37 °C for 1 hour. | ||
* Place 200 uL of the transformed cells on plates containing LB and the appropriate antibiotic. | * Place 200 uL of the transformed cells on plates containing LB and the appropriate antibiotic. | ||
- | *Incubate overnight at 37 °C. | + | * Incubate overnight at 37 °C. |
+ | </div> | ||
- | =Liquid Cultures | + | <div class="floatbox"> |
+ | <h1>Liquid Cultures</h1> | ||
- | + | Materials | |
* LB | * LB | ||
* Plated colonies of cells | * Plated colonies of cells | ||
- | *Antibiotic stock | + | * Antibiotic stock |
- | + | Procedure | |
* In a sterile environment, add 4 mL of LB to each falcon tube. | * In a sterile environment, add 4 mL of LB to each falcon tube. | ||
* Add the appropriate amount of antibiotic. | * Add the appropriate amount of antibiotic. | ||
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* Incubate overnight at 37 °C. | * Incubate overnight at 37 °C. | ||
- | = PCR | + | </div> |
- | + | ||
+ | <div class="floatbox"> | ||
+ | <h1>PCR</h1> | ||
+ | |||
+ | Mix | ||
*10ul Q solution | *10ul Q solution | ||
*5ul 10x buffer | *5ul 10x buffer | ||
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*30ul dH2O | *30ul dH2O | ||
- | =Minipreps | + | <div class="floatbox"> |
+ | <h1>Minipreps</h1> | ||
- | + | Materials | |
* Liquid culture | * Liquid culture | ||
* Miniprep kit (QIAprep Spin Miniprep Kit) | * Miniprep kit (QIAprep Spin Miniprep Kit) | ||
- | + | Procedure | |
* Centrifuge liquid culture of cells. | * Centrifuge liquid culture of cells. | ||
* Discard the supernatant. | * Discard the supernatant. | ||
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* Centrifuge for 1 minute and collect the flow-through. | * Centrifuge for 1 minute and collect the flow-through. | ||
- | + | </div> | |
- | = | + | <div class="floatbox"> |
+ | <h1>Ligations</h1> | ||
+ | |||
+ | Materials | ||
* Digested vector | * Digested vector | ||
* Digested insert | * Digested insert | ||
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* T4 DNA ligase buffer. | * T4 DNA ligase buffer. | ||
- | + | Procedure | |
* Mix these materials in the amounts determined by the reaction volume calculator. | * Mix these materials in the amounts determined by the reaction volume calculator. | ||
[[media:UC_Davis_Reaction_Volume_Calculator.xls]] | [[media:UC_Davis_Reaction_Volume_Calculator.xls]] | ||
+ | </html> |
Revision as of 07:20, 26 August 2011
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