Team:Calgary/Notebook/ProtocolManual/DNA

From 2014.igem.org

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<li>Centrifuge the subculture at max for 20 min at 4oC</li>
<li>Centrifuge the subculture at max for 20 min at 4oC</li>
<li>Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)</li>
<li>Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)</li>
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<li>Incubate at room temperature for 10 min</li>
<li>Incubate at room temperature for 10 min</li>
<li>Transform cells with DNA (1μL)</li>
<li>Transform cells with DNA (1μL)</li>
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</ol>
Store plates in -20oC
Store plates in -20oC
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<li>Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100μL</li>
<li>Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100μL</li>
<li>Resuspend, spread 50μL on antibiotic plate</li>
<li>Resuspend, spread 50μL on antibiotic plate</li>
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Grow overnight in incubator at 37°C
Grow overnight in incubator at 37°C
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<li>Carefully discard supernatant, dry pellet upside down</li>
<li>Carefully discard supernatant, dry pellet upside down</li>
<li>Resuspend in sterile ddH2O (~30 μL)</li>
<li>Resuspend in sterile ddH2O (~30 μL)</li>
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Deactivate enzymes: place insert tube on 82oC heatblock, 20 mins. Preform Antarctic phosphatase treatment on Vector tube
Deactivate enzymes: place insert tube on 82oC heatblock, 20 mins. Preform Antarctic phosphatase treatment on Vector tube
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<b>Antarctic Phosphatase Treatment</b>
<p>
<p>
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<b>Antarctic Phosphatase Treatment</b>
 
<ol type=1>
<ol type=1>
<li>To Vector tube from restriction digest, add 10x Antarctic Phosphatase Buffer (5 μL), ddH2O (9 μL), and Antarctice phosphatase (1 μL)</li>
<li>To Vector tube from restriction digest, add 10x Antarctic Phosphatase Buffer (5 μL), ddH2O (9 μL), and Antarctice phosphatase (1 μL)</li>
<li>Place tube in 37oC water bath, 30 min</li>
<li>Place tube in 37oC water bath, 30 min</li>
<li>Transfer to 82oC heatblock, 20min</li>
<li>Transfer to 82oC heatblock, 20min</li>
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<b>Ligation (Using KAPA Quick Ligase Kit)</b>
<p>
<p>
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<b>Ligation (Using KAPA Quick Ligase Kit)</b>
 
<ul>
<ul>
<li>10 μL Ligase Buffer</li>
<li>10 μL Ligase Buffer</li>
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<li>1:3 to 1:5 [Vector:Insert] molar ratio of Insert DNA (from digest)</li>
<li>1:3 to 1:5 [Vector:Insert] molar ratio of Insert DNA (from digest)</li>
<li>1 μL Enzyme (Quick Ligase)</li>
<li>1 μL Enzyme (Quick Ligase)</li>
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<li>ddH2O to bring total vol to 20 μL</li>
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<li>ddH2O to bring total vol to 20 μL</li></ul>
Wait 5 minutes after adding the Quick Ligase enzyme, then transform cells
Wait 5 minutes after adding the Quick Ligase enzyme, then transform cells
</p>
</p>

Revision as of 18:39, 10 October 2014

DNA Protocols

Preparing Chemically Competent E. coli Cells

  1. Inoculate 5-10mL LB with Top10 E. coli culture at 37oC shaking over night
  2. Subculture 1mL of bacteria into 50mL LB at 37oC shaking until OD600 is 0.4-0.6 (~2.5 hr)
  3. Centrifuge the subculture at max for 20 min at 4oC
  4. Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)

Rehydrating Registry DNA

  1. Add ddH2O (10μL) to appropriate well (will turn orange)/li>
  2. Incubate at room temperature for 10 min
  3. Transform cells with DNA (1μL)
Store plates in -20oC

Bacterial Transformation

  1. Thaw 100μL of competent cells on ice right before use (do not thaw completely)
  2. Add DNA (max 20μL) to cells, flick to mix, and ice for 30 min
  3. Heat shock 2 min at 42°C
  4. Ice 5 min
  5. Add 250μL LB medium to each tube
  6. Incubate 30-60 min at 37°C shaking (Kan is always 1h+)
  7. Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100μL
  8. Resuspend, spread 50μL on antibiotic plate
Grow overnight in incubator at 37°C

Plasmid Miniprep (E. coli)

  • Resuspension (P1): 50mM Tris HCl (pH 8), 10mM EDTA, 100μg/mL RNase A (keep on ice, 4°C)
  • Lysis (P2): 200mM NaOH, 1% SDS
  • Precipitation (P3): 3M K Acetate (pH 5.5)
  1. Transfer O/N to 2mL tube and pellet culture (spin at 14,000 rpm for 2 mins). Dump supernatant, and repeat for the rest of O/N.
  2. Discard supernatant, resuspend pellet in P1 (300μL)
  3. Quickly: Add P2 (300 μL), invert gently 2-3x. Add P3 (300 μL), invert gently 2-3x
  4. Centrifuge at 14,000 rpm, 10mins, room temp
  5. Without disturbing pellet, transfer supernatant to clean 1.5mL tube. Add isopropanol (650 μL). Wait 10 mins, room temp
  6. Spin at 14,000 rpm for 10 mins at 4oC
  7. Discard supernatant, add cold 70% etOH (500 μL)
  8. Centrifuge at 14,000 rpm for 5 mins at 4oC
  9. Carefully discard supernatant, dry pellet upside down
  10. Resuspend in sterile ddH2O (~30 μL)

Restriction Digest

Separately, into labeled tubes for Insert and Vector, add the following reagents:

  • 1μg DNA
  • 5μL of CutSmart Buffer
  • 0.5 to 1μL enzyme 1
  • 0.5 to 1μL enzyme 2
  • ddH2O to bring total vol to 50μL
Incubate tubes in 37oC water bath, 1 hr Deactivate enzymes: place insert tube on 82oC heatblock, 20 mins. Preform Antarctic phosphatase treatment on Vector tube

Antarctic Phosphatase Treatment

  1. To Vector tube from restriction digest, add 10x Antarctic Phosphatase Buffer (5 μL), ddH2O (9 μL), and Antarctice phosphatase (1 μL)
  2. Place tube in 37oC water bath, 30 min
  3. Transfer to 82oC heatblock, 20min

Ligation (Using KAPA Quick Ligase Kit)

  • 10 μL Ligase Buffer
  • 50ng Vector DNA (from digest)
  • 1:3 to 1:5 [Vector:Insert] molar ratio of Insert DNA (from digest)
  • 1 μL Enzyme (Quick Ligase)
  • ddH2O to bring total vol to 20 μL
Wait 5 minutes after adding the Quick Ligase enzyme, then transform cells

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