Difference between revisions of "Team:Michigan/Experiments"
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■ 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> | ■ 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> | ||
| − | <b>Procedure</b> | + | <b>Procedure</b><br> |
1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 µl of Buffer PB to 100 µl PCR sample (not including oil).<br> | 1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 µl of Buffer PB to 100 µl PCR sample (not including oil).<br> | ||
| − | 2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow<br> | + | 2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.<br> |
3. Place a QIAquick spin column in a provided 2 ml collection tube.<br> | 3. Place a QIAquick spin column in a provided 2 ml collection tube.<br> | ||
4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.<br> | 4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.<br> | ||
Revision as of 06:03, 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.
Procedure
1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 µl of Buffer PB to 100 µl PCR sample (not including oil).
2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
3. Place a QIAquick spin column in a provided 2 ml collection tube.
4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
5. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.
6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation. PCR Purification Spin Protocol
8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge. IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer volume, and 28 µl from 30 µl elution buffer. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel. Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and orange G) that facilitate estimation of DNA migration distance and optimization of agarose gel run time. Refer to Table 2 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.