Team:Glendale CC AZ/Protocols/AlkalineLysis

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== Alkaline Lysis Plasmid Miniprep Protocol ==
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Purpose: To isolate the plasmid DNA from transformed cells.  
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<h4>Glendale Community College Arizona<img
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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">
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<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>
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<a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/SurivalGrowth">Survival Growth  Assay</a></p>
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<a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/AlkalineLysis">Alkaline Lysis Plasmid Miniprep </a></p>
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<a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/RestrictionDigest">Restriction Digest</a></p>
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<a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/DNAIsolation">DNA Isolation</a></p>
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<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>
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<a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/Transformation">Transformation</a></p>
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==== Alkaline Lysis Plasmid Miniprep Protocol ====
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Purpose: To isolate the plasmid DNA from transformed cells.
 +
 
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==== Reagents: ====
    
    
Lysis solution (50mM glucose, 25mM TrisHCl pH 8, 10 mM EDTA)
Lysis solution (50mM glucose, 25mM TrisHCl pH 8, 10 mM EDTA)
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SDS/NaOH (prepare fresh)
SDS/NaOH (prepare fresh)
-
   -880ul dH2O
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   -880µl dH2O
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   -100ul 10% SDS
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   -100µl 10% SDS
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   -20ul 10M NaOh
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   -20µl 10M NaOh
Acetate solution
Acetate solution
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   -28.5mL dH2O
   -28.5mL dH2O
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== Materials: ==
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==== Materials: ====
-
-1 colony of transformed cells
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        -1 colony of transformed cells
         -5mL LB/chloramphenicol media
         -5mL LB/chloramphenicol media
         -Microcentrifuge
         -Microcentrifuge
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         -Micropipette
         -Micropipette
         -Disposable tips
         -Disposable tips
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         -200ul lysis solution
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         -200µl lysis solution
-
         -400ul SDS/NaOH (made fresh)
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         -400µl SDS/NaOH (made fresh)
-
         -300ul acetate solution
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         -300µl acetate solution
-
         -1000ul isopropanol
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         -1000µl isopropanol
-
         -400ul 70% ethanol
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         -400µl 70% ethanol
         -Kimwipes
         -Kimwipes
-
         -100 microliters TE buffer
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         -100µl TE buffer
-
+
-
== Procedure: ==
+
==== Procedure: ====
-
1. Inoculate 5ml of LB/antibiotic (chloramphenicol) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking.  
+
1. Inoculate 5ml of LB/antibiotic (chloramphenicol, or other) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking.  
2. Optional: Keep 2mL of the bacterial culture for indexing. Pipet 1.5mL of culture into a microcentrifuge tube, and microcentrifuge at 10,000 rpm for 1 minute. Discard the supernatant, and add the remaining 1.5mL of culture to the same microcentrifuge tube. Centrifuge the tube for 1 more minute at 10,000 rpm and discard supernatant one more time.
2. Optional: Keep 2mL of the bacterial culture for indexing. Pipet 1.5mL of culture into a microcentrifuge tube, and microcentrifuge at 10,000 rpm for 1 minute. Discard the supernatant, and add the remaining 1.5mL of culture to the same microcentrifuge tube. Centrifuge the tube for 1 more minute at 10,000 rpm and discard supernatant one more time.
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3. Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes.
3. Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes.
-
4. Add 400ul of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous.
+
4. Add 400µl of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous.
5. Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes.
5. Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes.
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11. Discard supernatant, being careful to avoid losing the pellet. Let rest at room temperature with the lid open or inverted on a Kimwipe to evaporate residual alcohol.
11. Discard supernatant, being careful to avoid losing the pellet. Let rest at room temperature with the lid open or inverted on a Kimwipe to evaporate residual alcohol.
-
12. Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55ul. Optional: Let pellet hydrate overnight.
+
12. Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55µl. Optional: Let pellet hydrate overnight.

Latest revision as of 02:35, 28 September 2013

iGEM

Glendale Community College ArizonaGCC

Protocols

iGEM Growth Curve Assay

NaCl Growth Curve Assay

Survival Growth Assay

Alkaline Lysis Plasmid Miniprep

Restriction Digest

DNA Isolation

Bioinformatics

Ligation

Transformation

Contents

Alkaline Lysis Plasmid Miniprep Protocol

Purpose: To isolate the plasmid DNA from transformed cells.

Reagents:

Lysis solution (50mM glucose, 25mM TrisHCl pH 8, 10 mM EDTA) 

  -9.0mL dH2O
  -0.25mL 1M TrisHCl pH 8
  -0.25mL 0.5 M Na2EDTA
  -0.50mL 20% glucose

SDS/NaOH (prepare fresh) 

  -880µl dH2O
  -100µl 10% SDS
  -20µl 10M NaOh

Acetate solution 

  -60mL 5M potassium acetate
  -11.5mL glacial acetic acid
  -28.5mL dH2O

Materials: 

       -1 colony of transformed cells
       -5mL LB/chloramphenicol media
       -Microcentrifuge
       -Microcentrifuge tubes
       -Micropipette
       -Disposable tips
       -200µl lysis solution
       -400µl SDS/NaOH (made fresh)
       -300µl acetate solution
       -1000µl isopropanol
       -400µl 70% ethanol
       -Kimwipes
       -100µl TE buffer

Procedure:

1. Inoculate 5ml of LB/antibiotic (chloramphenicol, or other) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking.

2. Optional: Keep 2mL of the bacterial culture for indexing. Pipet 1.5mL of culture into a microcentrifuge tube, and microcentrifuge at 10,000 rpm for 1 minute. Discard the supernatant, and add the remaining 1.5mL of culture to the same microcentrifuge tube. Centrifuge the tube for 1 more minute at 10,000 rpm and discard supernatant one more time.

3. Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes.

4. Add 400µl of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous.

5. Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes.

6. Centrifuge for 3 minutes at 10,000 rpm in microcentrifuge.

7. Decant supernatant into a clean microcentrifuge tube; avoid the loose, viscous pellet.

8. Add 1 volume of isopropanol and invert tube 3-6 times. Incubate at room temperature for 10 minutes to precipitate DNA.

9. Centrifuge at 10,000 rpm for 10 minutes. Orient hinge to the same direction in case pellet is hard to see.

10. Discard supernatant, removing residual liquid with pipet tip. Add 200-400ul 70% ethanol, agitate pellet gently and centrifuge at 10,000 rpm for 10 min to precipitate pellet.

11. Discard supernatant, being careful to avoid losing the pellet. Let rest at room temperature with the lid open or inverted on a Kimwipe to evaporate residual alcohol.

12. Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55µl. Optional: Let pellet hydrate overnight.

Retrieved from "http://2013.igem.org/Team:Glendale_CC_AZ/Protocols/AlkalineLysis"