Team:Calgary/Notebook/ProtocolManual/DNA
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- | {{Team:Calgary/ | + | {{Team:Calgary/Main}} |
<html> | <html> | ||
- | <section class="Content Text Color- | + | <section class="Content Text Color-Normal"> |
<h1>DNA Protocols</h1> | <h1>DNA Protocols</h1> | ||
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<p> | <p> | ||
<b>Rehydrating Registry DNA</b> | <b>Rehydrating Registry DNA</b> | ||
<ol type=1> | <ol type=1> | ||
- | <li>Add | + | <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 ( | + | <li>Transform cells with DNA (1 μL)</li></ol> |
- | </ol> | + | Store plates in -20 °C |
- | Store plates in - | + | |
</p> | </p> | ||
<p> | <p> | ||
<b>Bacterial Transformation</b> | <b>Bacterial Transformation</b> | ||
<ol type=1> | <ol type=1> | ||
- | <li>Thaw | + | <li>Thaw 100 μL of competent cells on ice right before use (do not thaw completely)</li> |
- | <li>Add DNA (max | + | <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 | + | <li>Heat shock 2 min at 42 °C</li> |
- | <li> | + | <li>Incubate on ice 5 min</li> |
- | <li>Add | + | <li>Add 250 μL LB medium to each tube</li> |
- | <li>Incubate 30-60 min at | + | <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 ~ | + | <li>Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100 μL</li> |
- | <li>Resuspend, spread | + | <li>Resuspend, spread 50 μL on antibiotic plate</li> |
- | < | + | <li>Grow overnight in incubator at 37°C</li> |
- | Grow overnight in incubator at 37°C | + | </ol></p> |
- | </p> | + | <p><b>Plasmid Miniprep (<i>E. coli</i>)</b></p> |
- | <p> | + | <p style="margin-bottom: 0;">Reagents: |
- | <b>Plasmid Miniprep (<i>E. coli</i>)</b> | + | <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>Resuspension (P1): | + | <li>Precipitation (P3): 3 M K Acetate (pH 5.5)</li> |
- | <li>Lysis (P2): | + | |
- | <li>Precipitation (P3): | + | |
</ul> | </ul> | ||
<ol type=1> | <ol type=1> | ||
- | <li>Transfer O/N to | + | <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 ( | + | <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, | + | <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 | + | <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 | + | <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 | + | <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> | ||
- | < | + | <p><b>Gel Extraction (EZNA by Promega)</b> |
- | < | + | |
<ul> | <ul> | ||
- | <li> | + | <li>Binding Buffer (XP2)</li> |
- | <li> | + | <li>SPW Wash Buffer (with ethanol)</li> |
- | <li> | + | <li>Transilluminator</li> |
- | + | ||
- | + | ||
</ul> | </ul> | ||
- | Incubate tubes in | + | |
- | + | <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> |
- | + | ||
<ol type=1> | <ol type=1> | ||
- | <li>To Vector tube from restriction digest, add 10x Antarctic Phosphatase Buffer (5 μL), | + | <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 | + | <li>Place tube in 37 °C water bath, 30 min</li> |
- | <li>Transfer to | + | <li>Transfer to 82 °C heatblock, 20min</li> |
</ol> | </ol> | ||
</p> | </p> | ||
- | <b>Ligation (Using KAPA | + | <p><b>Ligation (Using KAPA Rapid Ligase Kit)</b> |
- | + | ||
<ul> | <ul> | ||
<li>10 μL Ligase Buffer</li> | <li>10 μL Ligase Buffer</li> | ||
- | <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 ( | + | <li>1 μL Enzyme (Rapid Ligase)</li> |
- | <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 | 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
- Add ddH2O (10 μL) to appropriate well (will turn orange)
- Incubate at room temperature for 10 min
- Transform cells with DNA (1 μL)
Bacterial Transformation
- Thaw 100 μL of competent cells on ice right before use (do not thaw completely)
- Add DNA (max 20 μL) to cells, flick to mix, and incubate on ice for 30 min
- Heat shock 2 min at 42 °C
- Incubate on ice 5 min
- Add 250 μL LB medium to each tube
- Incubate 30-60 min at 37 °C shaking (If selecting on Kan incubation time is minimum 1 h)
- Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100 μL
- Resuspend, spread 50 μL on antibiotic plate
- 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)
- 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.
- Discard supernatant, resuspend pellet in P1 (300 μL)
- Quickly: Add P2 (300 μL), invert gently 2-3x. Add P3 (300 μL), invert gently 2-3x
- Centrifuge at 14,000 rpm, 10 mins, room temp
- Without disturbing pellet, transfer supernatant to clean 1.5mL tube. Add isopropanol (650 μL). Wait 10 mins, room temp
- Spin at 14,000 rpm for 10 mins at 4°C
- Discard supernatant, add cold 70% etOH (500 μL)
- Centrifuge at 14,000 rpm for 5 mins at 4°C
- Carefully discard supernatant, dry pellet upside down
- Resuspend in sterile ddH2O (~30 μL)
Plasmid Miniprep (GenElute Kit)
- Pellet cells from O/N culture
- Discard supernatant, and resuspend with Resuspension solution (200 μL)
- Add Lysis Solution (300 μL), immediately invert 6-8x (gently) until mixture becomes clear. Do not allow reaction to exceed 5 mins
- 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
- 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
- Transfer the clear lysate (from step 4) to the column, and spin at 13,000 rpm, 1 min. Discard flow through
- Add Wash Solution (750 μL) to column. Spin at 13,000 rpm, 1 min. Discard flow through and spin again at max, 2 mins
- 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
- Excise desired DNA bands from agarose gel using a transilluminator for visualization
- 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
- Heat tubes at 60 °C for 7 minutes or until the gel has fully dissolved. Vortex frequently
- Transfer the solution into a filter column placed within a 2 ml centrifuge tube. Do not exceed 700uL.
- Centrifuge at 14,000 RPM for 1 minute. Repeat until entire solution has been filtered.
- Discard solution and add 700 uL of SPW Wash Buffer to each column.
- Centrifuge at 14,00 RPM for 1 minute. Discard solution and centrifuge again to dry completely.
- 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
Antarctic Phosphatase Treatment
- To Vector tube from restriction digest, add 10x Antarctic Phosphatase Buffer (5 μL), ddH2O (9 μL), and Antarctice phosphatase (1 μL)
- Place tube in 37 °C water bath, 30 min
- 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