Difference between revisions of "Team:ULaVerne Collab/notebook"

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10. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.<BR>
 
10. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.<BR>
  
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<h2 class="content-title">Ligation</h2>
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</section>
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<h2 class="content-title">QIAEX II Gel Extraction Kit Protocol</h2>
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</section>
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<h2 class="content-title">QIAquick PCR Purification Kit</h2>
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</section>
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<div class="more-content">
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<h2 class="content-title">CHLAMYDOMONAS PROTOCOLS</h2>
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<h2 class="content-title">Transformation Protocol (E. coli)</h2>
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1. Pre-chill microcentrifuge tube and thaw competent cells on ice. <BR>
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2. Add 50uL of competent cells into the chilled microfuge tube.  <BR>
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3. Add 4uL of DNA into the microcentrifuge tube. <BR>
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4. Incubate on ice on 30 minutes. <BR>
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5. Heat shock at 42 degrees Celsius for 45 sec. <BR>
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6. Incubate cells on ice for 2 minutes. <BR>
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7. Add 200uL of SOC media into the microcentrifuge tube. <BR>
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8.Incubate and shake at 37 degree Celsius for 1-2 hours. <BR>
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9. Pipette 200uL of cells into LB plate with ampicillin resistance. <BR>
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10. Incubate plate overnight at 37 degrees Celsius.  <BR>
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<h2 class="content-title">Miniprep</h2>
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1. Transfer 1.5mL of overnight bacterial cultures from conicol tubes to microcentrifuge tubes. Centrifuge at 8000rpm for 3 minutes at room temperature. Discard supernatant and repeat until all of the overnight bacterial culture is suspended. <BR>
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2. Resuspend pelleted bacterial cells in 250uL buffer P1.<BR>
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3. Add 250uL buffer P2 and mix by inverting 4-6 times. <BR>
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4. Add 350uL buffer N3 and mix by inverting.<BR>
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5. Centrifuge for 10 minutes at 1300rpm. <BR>
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6. Pipette the supernatant from step 5 to the QIAprep spin column. Centrifuge for 1 minute and discard flow through.<BR>
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7. Wash the QIAprep spin column by adding 750uL buffer PE. Centrifuge for 1 minute and discard flow through. <BR>
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8. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. Add 50uL nuclease free water to the center of the QIAprep spin column. Let it stand for 1 minute. Centrifuge for 1 minute. <BR>
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</section>
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<section class="more grid">
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<div class="more-content">
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<h2 class="content-title">Digestion</h2>
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<div id="bodyText">
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1. Thaw all enzymes need and aliquot appropriate amounts. <BR>
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2. Pipette the amount of nuclease free water needed to make 500ug of DNA. (500 ug/Concentration. Then take that amount and subtract it from 50uL to get the amount of water needed) <BR>
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3. Add appropriate amount of DNA into the nuclease free water. <BR>
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4. Add 5uL of NEB Buffer to each tube. <BR>
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5. Add 1uL of the first enzyme to each tube. (BsrG1) <BR>
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6. Add 1uL of the second enzyme to each tube. (BamHI) <BR>
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7. Add 1uL of SAP to vector tube.<BR>
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8. Agitate each solution to mix well.<BR>
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9. Incubate parts at 37 degrees Celsius for 15 minutes. Incubate vector at 37 degrees Celsius for 30 minutes.<BR>
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10. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.<BR>
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</div>
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</div>
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</section>
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<section class="more grid">
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<div class="more-content">
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<h2 class="content-title">Ligation</h2>
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<div id="bodyText">
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</div>
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</div>
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</section>
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<section class="more grid">
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<div class="more-content">
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<h2 class="content-title">QIAEX II Gel Extraction Kit Protocol</h2>
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<div id="bodyText">
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</div>
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</div>
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</section>
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<section class="more grid">
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<div class="more-content">
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<h2 class="content-title">QIAquick PCR Purification Kit</h2>
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Revision as of 17:05, 1 November 2017

MAIN

HOME
TEAM
ATTRIBUTIONS
PROJECT
PARTS
MODEL
PROTOCOLS
COLLABORATIONS
COMMUNITY ENGAGEMENT
APPLIED DESIGN
INTEGRATED PRACTICES
AWARDS
SAFETY

PROTOCOLS

Transformation Protocol (E. coli)

1. Pre-chill microcentrifuge tube and thaw competent cells on ice.
2. Add 50uL of competent cells into the chilled microfuge tube.
3. Add 4uL of DNA into the microcentrifuge tube.
4. Incubate on ice on 30 minutes.
5. Heat shock at 42 degrees Celsius for 45 sec.
6. Incubate cells on ice for 2 minutes.
7. Add 200uL of SOC media into the microcentrifuge tube.
8.Incubate and shake at 37 degree Celsius for 1-2 hours.
9. Pipette 200uL of cells into LB plate with ampicillin resistance.
10. Incubate plate overnight at 37 degrees Celsius.

Miniprep

1. Transfer 1.5mL of overnight bacterial cultures from conicol tubes to microcentrifuge tubes. Centrifuge at 8000rpm for 3 minutes at room temperature. Discard supernatant and repeat until all of the overnight bacterial culture is suspended.
2. Resuspend pelleted bacterial cells in 250uL buffer P1.
3. Add 250uL buffer P2 and mix by inverting 4-6 times.
4. Add 350uL buffer N3 and mix by inverting.
5. Centrifuge for 10 minutes at 1300rpm.
6. Pipette the supernatant from step 5 to the QIAprep spin column. Centrifuge for 1 minute and discard flow through.
7. Wash the QIAprep spin column by adding 750uL buffer PE. Centrifuge for 1 minute and discard flow through.
8. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. Add 50uL nuclease free water to the center of the QIAprep spin column. Let it stand for 1 minute. Centrifuge for 1 minute.

Digestion

1. Thaw all enzymes need and aliquot appropriate amounts.
2. Pipette the amount of nuclease free water needed to make 500ug of DNA. (500 ug/Concentration. Then take that amount and subtract it from 50uL to get the amount of water needed)
3. Add appropriate amount of DNA into the nuclease free water.
4. Add 5uL of NEB Buffer to each tube.
5. Add 1uL of the first enzyme to each tube. (BsrG1)
6. Add 1uL of the second enzyme to each tube. (BamHI)
7. Add 1uL of SAP to vector tube.
8. Agitate each solution to mix well.
9. Incubate parts at 37 degrees Celsius for 15 minutes. Incubate vector at 37 degrees Celsius for 30 minutes.
10. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.

Ligation

QIAEX II Gel Extraction Kit Protocol

QIAquick PCR Purification Kit

CHLAMYDOMONAS PROTOCOLS

Transformation Protocol (E. coli)

1. Pre-chill microcentrifuge tube and thaw competent cells on ice.
2. Add 50uL of competent cells into the chilled microfuge tube.
3. Add 4uL of DNA into the microcentrifuge tube.
4. Incubate on ice on 30 minutes.
5. Heat shock at 42 degrees Celsius for 45 sec.
6. Incubate cells on ice for 2 minutes.
7. Add 200uL of SOC media into the microcentrifuge tube.
8.Incubate and shake at 37 degree Celsius for 1-2 hours.
9. Pipette 200uL of cells into LB plate with ampicillin resistance.
10. Incubate plate overnight at 37 degrees Celsius.

Miniprep

1. Transfer 1.5mL of overnight bacterial cultures from conicol tubes to microcentrifuge tubes. Centrifuge at 8000rpm for 3 minutes at room temperature. Discard supernatant and repeat until all of the overnight bacterial culture is suspended.
2. Resuspend pelleted bacterial cells in 250uL buffer P1.
3. Add 250uL buffer P2 and mix by inverting 4-6 times.
4. Add 350uL buffer N3 and mix by inverting.
5. Centrifuge for 10 minutes at 1300rpm.
6. Pipette the supernatant from step 5 to the QIAprep spin column. Centrifuge for 1 minute and discard flow through.
7. Wash the QIAprep spin column by adding 750uL buffer PE. Centrifuge for 1 minute and discard flow through.
8. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. Add 50uL nuclease free water to the center of the QIAprep spin column. Let it stand for 1 minute. Centrifuge for 1 minute.

Digestion

1. Thaw all enzymes need and aliquot appropriate amounts.
2. Pipette the amount of nuclease free water needed to make 500ug of DNA. (500 ug/Concentration. Then take that amount and subtract it from 50uL to get the amount of water needed)
3. Add appropriate amount of DNA into the nuclease free water.
4. Add 5uL of NEB Buffer to each tube.
5. Add 1uL of the first enzyme to each tube. (BsrG1)
6. Add 1uL of the second enzyme to each tube. (BamHI)
7. Add 1uL of SAP to vector tube.
8. Agitate each solution to mix well.
9. Incubate parts at 37 degrees Celsius for 15 minutes. Incubate vector at 37 degrees Celsius for 30 minutes.
10. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.

Ligation

QIAEX II Gel Extraction Kit Protocol

QIAquick PCR Purification Kit