Difference between revisions of "Team:Michigan/Experiments"
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1. Set up the following reaction in a microcentrifuge tube on ice.<br> | 1. Set up the following reaction in a microcentrifuge tube on ice.<br> | ||
2. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) <br> | 2. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) <br> | ||
| − | |||
<img src="https://static.igem.org/mediawiki/2015/6/67/Experiment1.png" alt="Experiment 1" style="width:75%;height:75%;"><br> | <img src="https://static.igem.org/mediawiki/2015/6/67/Experiment1.png" alt="Experiment 1" style="width:75%;height:75%;"><br> | ||
* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.<br><br> | * The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.<br><br> | ||
| Line 69: | Line 68: | ||
5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours<br> | 5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours<br> | ||
6. Heat inactivate at 65°C for 10 minutes.<br> | 6. Heat inactivate at 65°C for 10 minutes.<br> | ||
| − | 7. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.<br> | + | 7. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.<br><br> |
| + | <h4>High Efficiency Transformation Protocol</h4><br> | ||
| + | 1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.<br> | ||
| + | 2. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.<br> | ||
| + | 3. Add 1-5 µl containing 100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.<br> | ||
| + | 4. Place the mixture on ice for 30 minutes. Do not mix.<br> | ||
| + | 5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.<br> | ||
| + | 6. Place on ice for 5 minutes. Do not mix.<br> | ||
| + | 7. Pipette 950 µl of room temperature SOC into the mixture.<br> | ||
| + | 8. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.<br> | ||
| + | 9. Warm selection plates to 37°C.<br> | ||
| + | 10. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.<br> | ||
| + | 11. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.<br> | ||
| + | <br><br> | ||
| + | <h4>QIAquick PCR Purification Kit Protocol using a microcentrifuge</h4><br> | ||
| + | This protocol is designed to purify single- or double-stranded DNA fragments from PCR and other enzymatic reactions. Fragments ranging from 100 bp to 10 kb are purified from primers, nucleotides, polymerases, and salts using QIAquick spin columns in a microcentrifuge.<br><br> | ||
| + | |||
| + | Important points before starting:<br> | ||
| + | ■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).<br> | ||
| + | ■ All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional tabletop microcentrifuge at room temperature.<br> | ||
| + | ■ Add 1:250 volume pH indicator I to Buffer PB (i.e., add 120 µl pH indicator I to 30 ml Buffer PB or add 600 µl pH indicator I to 150 ml Buffer PB). The yellow color of Buffer PB with pH indicator I indicates a pH of #7.5.<br> | ||
| + | ■ Add pH indicator I to entire buffer contents. Do not add pH indicator I to buffer aliquots.<br> | ||
| + | ■ If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I.<br><br><br> | ||
| + | |||
| + | <h4>QIAquick Gel Extraction Kit Protocol using a microcentrifuge</h4> | ||
| + | <h4>Phenol | Chloroform Extraction</h4> | ||
| + | <h4>Ethanol Precipitation</h4> | ||
| + | <h4>PURExpress® In Vitro Protein Synthesis</h4> | ||
| + | <h4>Plasmid DNA Purification using the QIAprep Spin Miniprep Kit</h4> | ||
| + | <h4>LB medium (1L liquid)</h4> | ||
| + | <h4>Setting up a Double Digestion </h4> | ||
</html> | </html> | ||
Revision as of 05:51, 20 November 2015
Experiments and Protocols
Ligation Protocol with T4 DNA Ligase
1. Set up the following reaction in a microcentrifuge tube on ice.
2. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.)
* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
3. Gently mix the reaction by pipetting up and down and microfuge briefly.
4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours
6. Heat inactivate at 65°C for 10 minutes.
7. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
High Efficiency Transformation Protocol
1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
2. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
3. Add 1-5 µl containing 100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
6. Place on ice for 5 minutes. Do not mix.
7. Pipette 950 µl of room temperature SOC into the mixture.
8. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
9. Warm selection plates to 37°C.
10. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
11. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
QIAquick PCR Purification Kit Protocol using a microcentrifuge
This protocol is designed to purify single- or double-stranded DNA fragments from PCR and other enzymatic reactions. Fragments ranging from 100 bp to 10 kb are purified from primers, nucleotides, polymerases, and salts using QIAquick spin columns in a microcentrifuge.
Important points before starting:
■ Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
■ All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional tabletop microcentrifuge at room temperature.
■ Add 1:250 volume pH indicator I to Buffer PB (i.e., add 120 µl pH indicator I to 30 ml Buffer PB or add 600 µl pH indicator I to 150 ml Buffer PB). The yellow color of Buffer PB with pH indicator I indicates a pH of #7.5.
■ Add pH indicator I to entire buffer contents. Do not add pH indicator I to buffer aliquots.
■ If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I.