Team:Calgary/Notebook/ProtocolManual/DNA

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<section class="Content Text Color-Dark">
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<section class="Content Text Color-Normal">
<h1>DNA Protocols</h1>
<h1>DNA Protocols</h1>
 +
<p>
<p>
-
<b>Preparing Chemically Competent <i>E. coli</i> Cells</b>
+
<b>Rehydrating Registry DNA</b>
<ol type=1>
<ol type=1>
-
<li>Inoculate 5-10mL LB with Top10 E. coli culture at 37oC shaking over night</li>
+
<li>Add ddH<sub>2</sub>O (10 μL) to appropriate well (will turn orange)</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>Incubate at room temperature for 10 min</li>
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<li>Centrifuge the subculture at max for 20 min at 4oC</li>
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<li>Transform cells with DNA (1 μL)</li></ol>
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<li>Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)</li>
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Store plates in -20 °C
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</ul>
+
</p>
</p>
<p>
<p>
-
<b>Rehydrating Registry DNA</b>
+
<b>Bacterial Transformation</b>
<ol type=1>
<ol type=1>
-
<li>Add ddH2O (10μL) to appropriate well (will turn orange)/li>
+
<li>Thaw 100 μL of competent cells on ice right before use (do not thaw completely)</li>
-
<li>Incubate at room temperature for 10 min</li>
+
<li>Add DNA (max 20 μL) to cells, flick to mix, and incubate on ice for 30 min</li>
-
<li>Transform cells with DNA (1μL)</li>
+
<li>Heat shock 2 min at 42 °C</li>
 +
<li>Incubate on ice 5 min</li>
 +
<li>Add 250 μL LB medium to each tube</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 ~100 μL</li>
 +
<li>Resuspend, spread 50 μL on antibiotic plate</li>
 +
<li>Grow overnight in incubator at 37°C</li>
 +
</ol></p>
 +
<p><b>Plasmid Miniprep (<i>E. coli</i>)</b></p>
 +
<p style="margin-bottom: 0;">Reagents:
 +
<ul><li>Resuspension (P1): 50 mM Tris HCl (pH 8), 10 mM EDTA, 100 μg/mL RNase A (keep on ice, 4 °C)</li>
 +
<li>Lysis (P2): 200 mM NaOH, 1% SDS</li>
 +
<li>Precipitation (P3): 3 M K Acetate (pH 5.5)</li>
</ul>
</ul>
-
Store plates in -20oC
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<ol type=1>
 +
<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 (300 μL)</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, 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 4°C</li>
 +
<li>Discard supernatant, add cold 70% etOH (500 μL)</li>
 +
<li>Centrifuge at 14,000 rpm for 5 mins at 4°C</li>
 +
<li>Carefully discard supernatant, dry pellet upside down</li>
 +
<li>Resuspend in sterile ddH<sub>2</sub>O (~30 μL)</li>
 +
</ol>
</p>
</p>
<p>
<p>
-
<b>Bacterial Transformation</b>
+
<b>Plasmid Miniprep (GenElute Kit)</b>
<ol type=1>
<ol type=1>
-
<li>Thaw 100μL of competent cells on ice right before use (do not thaw completely)</li>
+
<li>Pellet cells from O/N culture</li>
-
<li>Add DNA (max 20μL) to cells, flick to mix, and ice for 30 min</li>
+
<li>Discard supernatant, and resuspend with Resuspension solution (200 μL)</li>
-
<li>Heat shock 2 min at 42°C</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>Ice 5 min</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>Add 250μL LB medium to each tube</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>Incubate 30-60 min at 37°C shaking (Kan is always 1h+)</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>Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100μL</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>Resuspend, spread 50μL on antibiotic plate</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>
 +
</p>
 +
 
 +
<p><b>Gel Extraction (EZNA by Promega)</b>
 +
<ul>
 +
<li>Binding Buffer (XP2)</li>
 +
<li>SPW Wash Buffer (with ethanol)</li>
 +
<li>Transilluminator</li>
</ul>
</ul>
-
Grow overnight in incubator at 37°C
+
 
 +
<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>
 +
 
 +
<ol type=1>
 +
<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 37 °C water bath, 30 min</li>
 +
<li>Transfer to 82 °C heatblock, 20min</li>
 +
</ol>
 +
</p>
 +
 
 +
<p><b>Ligation (Using KAPA Rapid Ligase Kit)</b>
 +
 
 +
<ul>
 +
<li>10 μL Ligase Buffer</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 μL Enzyme (Rapid Ligase)</li>
 +
<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
 +
</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