Team:Calgary/Notebook/ProtocolManual/DNA

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<section class="Content Text Color-Normal">
<h1>DNA Protocols</h1>
<h1>DNA Protocols</h1>
-
<p>
+
 
-
<b>Preparing Chemically Competent <i>E. coli</i> Cells</b>
+
-
<ol type=1>
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-
<li>Inoculate 5-10mL LB with Top10 E. coli culture at 37oC shaking over night</li>
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-
<li>Subculture 1mL of bacteria into 50mL LB at 37oC shaking until OD<sub>600</sub> is 0.4-0.6 (~2.5 hr)</li>
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-
<li>Centrifuge the subculture at max for 20 min at 4oC</li>
+
-
<li>Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)</li>
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-
</ol>
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</p>
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<p>
<p>
<b>Rehydrating Registry DNA</b>
<b>Rehydrating Registry DNA</b>
<ol type=1>
<ol type=1>
-
<li>Add ddH2O (10μL) to appropriate well (will turn orange)</li>
+
<li>Add ddH<sub>2</sub>O (10 μL) to appropriate well (will turn orange)</li>
<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 (1 μL)</li></ol>
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</ol>
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Store plates in -20 °C
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Store plates in -20oC
+
</p>
</p>
<p>
<p>
<b>Bacterial Transformation</b>
<b>Bacterial Transformation</b>
<ol type=1>
<ol type=1>
-
<li>Thaw 100μL of competent cells on ice right before use (do not thaw completely)</li>
+
<li>Thaw 100 μL of competent cells on ice right before use (do not thaw completely)</li>
-
<li>Add DNA (max 20μL) to cells, flick to mix, and ice for 30 min</li>
+
<li>Add DNA (max 20 μL) to cells, flick to mix, and incubate on ice for 30 min</li>
-
<li>Heat shock 2 min at 42°C</li>
+
<li>Heat shock 2 min at 42 °C</li>
-
<li>Ice 5 min</li>
+
<li>Incubate on ice 5 min</li>
-
<li>Add 250μL LB medium to each tube</li>
+
<li>Add 250 μL LB medium to each tube</li>
-
<li>Incubate 30-60 min at 37°C shaking (Kan is always 1h+)</li>
+
<li>Incubate 30-60 min at 37 °C shaking (If selecting on Kan incubation time is minimum 1 h)</li>
-
<li>Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100μL</li>
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<li>Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100 μL</li>
-
<li>Resuspend, spread 50μL on antibiotic plate</li>
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<li>Resuspend, spread 50 μL on antibiotic plate</li>
-
</ol>
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<li>Grow overnight in incubator at 37°C</li>
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Grow overnight in incubator at 37°C
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</ol></p>
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</p>
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<p><b>Plasmid Miniprep (<i>E. coli</i>)</b></p>
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<p>
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<p style="margin-bottom: 0;">Reagents:
-
<b>Plasmid Miniprep (<i>E. coli</i>)</b>
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<ul><li>Resuspension (P1): 50 mM Tris HCl (pH 8), 10 mM EDTA, 100 μg/mL RNase A (keep on ice, 4 °C)</li>
-
<ul>
+
<li>Lysis (P2): 200 mM NaOH, 1% SDS</li>
-
<li>Resuspension (P1): 50mM Tris HCl (pH 8), 10mM EDTA, 100μg/mL RNase A (keep on ice, 4°C)</li>
+
<li>Precipitation (P3): 3 M K Acetate (pH 5.5)</li>
-
<li>Lysis (P2): 200mM NaOH, 1% SDS</li>
+
-
<li>Precipitation (P3): 3M K Acetate (pH 5.5)</li>
+
</ul>
</ul>
<ol type=1>
<ol type=1>
-
<li>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. </li>
+
<li>Transfer O/N to 2 mL tube and pellet culture (spin at 14,000 rpm for 2 mins). Dump supernatant, and repeat for the rest of O/N. </li>
-
<li>Discard supernatant, resuspend pellet in P1 (300μL)</li>
+
<li>Discard supernatant, resuspend pellet in P1 (300 μL)</li>
<li>Quickly: Add P2 (300 μL), invert gently 2-3x. Add P3 (300 μL), invert gently 2-3x</li>
<li>Quickly: Add P2 (300 μL), invert gently 2-3x. Add P3 (300 μL), invert gently 2-3x</li>
-
<li>Centrifuge at 14,000 rpm, 10mins, room temp</li>
+
<li>Centrifuge at 14,000 rpm, 10 mins, room temp</li>
<li>Without disturbing pellet, transfer supernatant to clean 1.5mL tube. Add isopropanol (650 μL). Wait 10 mins, room temp</li>
<li>Without disturbing pellet, transfer supernatant to clean 1.5mL tube. Add isopropanol (650 μL). Wait 10 mins, room temp</li>
-
<li>Spin at 14,000 rpm for 10 mins at 4oC</li>
+
<li>Spin at 14,000 rpm for 10 mins at 4°C</li>
<li>Discard supernatant, add cold 70% etOH (500 μL)</li>
<li>Discard supernatant, add cold 70% etOH (500 μL)</li>
-
<li>Centrifuge at 14,000 rpm for 5 mins at 4oC</li>
+
<li>Centrifuge at 14,000 rpm for 5 mins at 4°C</li>
<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 ddH<sub>2</sub>O (~30 μL)</li>
 +
</ol>
 +
</p>
 +
<p>
 +
<b>Plasmid Miniprep (GenElute Kit)</b>
 +
<ol type=1>
 +
<li>Pellet cells from O/N culture</li>
 +
<li>Discard supernatant, and resuspend with Resuspension solution (200 μL)</li>
 +
<li>Add Lysis Solution (300 μL), immediately invert 6-8x (gently) until mixture becomes clear. Do not allow reaction to exceed 5 mins</li>
 +
<li>Add Neutralization Solution (350 μL), invert 4-6x (gently). Pellet in centrifuge at max, 10 mins. If supernatant contains a large amount of floating particles, respin</li>
 +
<li>Meanwhile, prepare binding column. Insert column into a microcentrifuge tube and add Column Prep Solution (500 μL). Spin at 13,000 rpm for 1 min, Discard flow through</li>
 +
<li>Transfer the clear lysate (from step 4) to the column, and spin at 13,000 rpm, 1 min. Discard flow through</li>
 +
<li>Add Wash Solution (750 μL) to column. Spin at 13,000 rpm, 1 min. Discard flow through and spin again at max, 2 mins</li>
 +
<li>Transfer column a fresh collection tube and add ddH2O (~50 μL). Let sit 2 mins. Spin at 13,000 rpm, 1 min. Sample now ready for nano-drop</li>
</ol>
</ol>
</p>
</p>
-
<b>Restriction Digest</b>
+
<p><b>Gel Extraction (EZNA by Promega)</b>
-
<p>Separately, into labeled tubes for Insert and Vector, add the following reagents:
+
<ul>
<ul>
-
<li>1μg DNA</li>
+
<li>Binding Buffer (XP2)</li>
-
<li>5μL of CutSmart Buffer</li>
+
<li>SPW Wash Buffer (with ethanol)</li>
-
<li>0.5 to 1μL enzyme 1</li>
+
<li>Transilluminator</li>
-
<li>0.5 to 1μL enzyme 2</li>
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-
<li>ddH2O to bring total vol to 50μL</li>
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</ul>
</ul>
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Incubate tubes in 37oC water bath, 1 hr
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-
Deactivate enzymes: place insert tube on 82oC heatblock, 20 mins. Preform Antarctic phosphatase treatment on Vector tube
+
<ol>
 +
<li>Excise desired DNA bands from agarose gel using a transilluminator for visualization</li>
 +
<li>Place each band into a 1.5 ml centrifuge tube and add one volume of Binding Buffer (XP2). Assume the density of agarose gel to be 1 g/1ml</li>
 +
<li>Heat tubes at 60 °C for 7 minutes or until the gel has fully dissolved. Vortex frequently</li>
 +
<li>Transfer the solution into a filter column placed within a 2 ml centrifuge tube. Do not exceed 700uL.</li>
 +
<li>Centrifuge at 14,000 RPM for 1 minute. Repeat until entire solution has been filtered.</li>
 +
<li>Discard solution and add 700 uL of SPW Wash Buffer to each column.</li>
 +
<li>Centrifuge at 14,00 RPM for 1 minute. Discard solution and centrifuge again to dry completely.</li>
 +
<li>Transfer columns two new 2 ml centrifuge tubes. Soak columns with 30-50 uL of water for 1 minute before eluting with 14,000 RPM centrifugation.</li>
 +
</ol></p>
 +
 +
<p><b>Restriction Digest</b></p>
 +
Separately, into labeled tubes for Insert and Vector, add the following reagents:
 +
<ul>
 +
<li>1 μg DNA</li>
 +
<li>5 μL of CutSmart Buffer</li>
 +
<li>0.5 to 1 μL enzyme 1</li>
 +
<li>0.5 to 1 μL enzyme 2</li>
 +
<li>ddH<sub>2</sub>O to bring total volume to 50 μL</li>
 +
</ul>
 +
Incubate tubes in 37 °C water bath for 1 hr. To deactivate enzymes: heat shock by incubating at 82 °C for 20 mins. Preform Antarctic phosphatase treatment on Vector tube
</p>
</p>
-
<b>Antarctic Phosphatase Treatment</b>
+
<p><b>Antarctic Phosphatase Treatment</b>
-
<p>
+
 
<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), ddH<sub>2</sub>O (9 μL), and Antarctice phosphatase (1 μL)</li>
-
<li>Place tube in 37oC water bath, 30 min</li>
+
<li>Place tube in 37 °C water bath, 30 min</li>
-
<li>Transfer to 82oC heatblock, 20min</li>
+
<li>Transfer to 82 °C heatblock, 20min</li>
</ol>
</ol>
</p>
</p>
-
<b>Ligation (Using KAPA Quick Ligase Kit)</b>
+
<p><b>Ligation (Using KAPA Rapid Ligase Kit)</b>
-
<p>
+
 
<ul>
<ul>
<li>10 μL Ligase Buffer</li>
<li>10 μL Ligase Buffer</li>
-
<li>50ng Vector DNA (from digest)</li>
+
<li>50 ng Vector DNA (from digest)</li>
<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 (Rapid Ligase)</li>
-
<li>ddH2O to bring total vol to 20 μL</li></ul>
+
<li>ddH<sub>2</sub>O to bring total volume 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>

Latest revision as of 02:30, 18 October 2014

DNA Protocols

Rehydrating Registry DNA

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

Bacterial Transformation

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

Plasmid Miniprep (E. coli)

Reagents:

  • Resuspension (P1): 50 mM Tris HCl (pH 8), 10 mM EDTA, 100 μg/mL RNase A (keep on ice, 4 °C)
  • Lysis (P2): 200 mM NaOH, 1% SDS
  • Precipitation (P3): 3 M K Acetate (pH 5.5)
  1. Transfer O/N to 2 mL 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 (300 μL)
  3. Quickly: Add P2 (300 μL), invert gently 2-3x. Add P3 (300 μL), invert gently 2-3x
  4. Centrifuge at 14,000 rpm, 10 mins, 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 4°C
  7. Discard supernatant, add cold 70% etOH (500 μL)
  8. Centrifuge at 14,000 rpm for 5 mins at 4°C
  9. Carefully discard supernatant, dry pellet upside down
  10. Resuspend in sterile ddH2O (~30 μL)

Plasmid Miniprep (GenElute Kit)

  1. Pellet cells from O/N culture
  2. Discard supernatant, and resuspend with Resuspension solution (200 μL)
  3. Add Lysis Solution (300 μL), immediately invert 6-8x (gently) until mixture becomes clear. Do not allow reaction to exceed 5 mins
  4. Add Neutralization Solution (350 μL), invert 4-6x (gently). Pellet in centrifuge at max, 10 mins. If supernatant contains a large amount of floating particles, respin
  5. Meanwhile, prepare binding column. Insert column into a microcentrifuge tube and add Column Prep Solution (500 μL). Spin at 13,000 rpm for 1 min, Discard flow through
  6. Transfer the clear lysate (from step 4) to the column, and spin at 13,000 rpm, 1 min. Discard flow through
  7. Add Wash Solution (750 μL) to column. Spin at 13,000 rpm, 1 min. Discard flow through and spin again at max, 2 mins
  8. Transfer column a fresh collection tube and add ddH2O (~50 μL). Let sit 2 mins. Spin at 13,000 rpm, 1 min. Sample now ready for nano-drop

Gel Extraction (EZNA by Promega)

  • Binding Buffer (XP2)
  • SPW Wash Buffer (with ethanol)
  • Transilluminator
  1. Excise desired DNA bands from agarose gel using a transilluminator for visualization
  2. Place each band into a 1.5 ml centrifuge tube and add one volume of Binding Buffer (XP2). Assume the density of agarose gel to be 1 g/1ml
  3. Heat tubes at 60 °C for 7 minutes or until the gel has fully dissolved. Vortex frequently
  4. Transfer the solution into a filter column placed within a 2 ml centrifuge tube. Do not exceed 700uL.
  5. Centrifuge at 14,000 RPM for 1 minute. Repeat until entire solution has been filtered.
  6. Discard solution and add 700 uL of SPW Wash Buffer to each column.
  7. Centrifuge at 14,00 RPM for 1 minute. Discard solution and centrifuge again to dry completely.
  8. Transfer columns two new 2 ml centrifuge tubes. Soak columns with 30-50 uL of water for 1 minute before eluting with 14,000 RPM centrifugation.

Restriction Digest

Separately, into labeled tubes for Insert and Vector, add the following reagents:
  • 1 μg DNA
  • 5 μL of CutSmart Buffer
  • 0.5 to 1 μL enzyme 1
  • 0.5 to 1 μL enzyme 2
  • ddH2O to bring total volume to 50 μL
Incubate tubes in 37 °C water bath for 1 hr. To deactivate enzymes: heat shock by incubating at 82 °C for 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 37 °C water bath, 30 min
  3. Transfer to 82 °C heatblock, 20min

Ligation (Using KAPA Rapid Ligase Kit)

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