代做BIO2101 Comprehensive Biology Laboratory Exercise 5代写数据结构语言程序
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Exercise 5: Polymerase chain reaction (PCR) replication of β-galactosidase gene LacZ and gel electrophoresis verification
Purpose:
1. Understand the principle of DNA fragment replication via PCR methods and practice it.
2. Perform. DNA gel electrophoresis to identify base pair number of a certain DNA fragment.
I. Introduction:
Part 1: polymerase chain reaction (PCR) replication of LacZ
In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase. PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence).
A standard polymerase chain reaction (PCR) setup consists of following steps:
1. Designing Primers
Designing appropriate primers is essential to the successful outcome of a PCR experiment. When designing a set of primers to a specific region of DNA desired for amplification, one primer should anneal to the plus strand, which by convention is oriented in the 5' → 3' direction (also known as the sense or nontemplate strand) and the other primer should complement the minus strand, which is oriented in the 3' → 5' direction (antisense or template strand).
Below is a list of characteristics that should be considered when designing primers.
1) Primer length should be 15-30 nucleotide residues (bases).
2) Optimal G-C content should range between 40-60%.
3) The 3' end of primers should contain a G or C in order to clamp the primer and prevent "breathing" of ends, increasing priming efficiency. DNA "breathing" occurs when ends do not stay annealed but fray or split apart. The three hydrogen bonds in GC pairs help prevent breathing but also increase the melting temperature of the primers.
4) The 3' ends of a primer set, which includes a plus strand primer and a
minus strand primer, should not be complementary to each other, nor can the 3' end of a single primer be complementary to other sequences in the primer. These two scenarios result in formation of primer dimers and hairpin loop structures, respectively.
5) Optimal melting temperatures (Tm) for primers range between 52-58 °C, although the range can be expanded to 45-65 °C. The final Tm for both primers should differ by no more than 5 °C.
6) Di-nucleotide repeats (e.g., GCGCGCGCGC or ATATATATAT) or single base runs (e.g., AAAAA or CCCCC) should be avoided as they can cause slipping along the primed segment of DNA and or hairpin loop structures to form. If unavoidable due to nature of the DNA template, then only include repeats or single base runs with a maximum of 4 bases.
Notes:
l There are many computer programs designed to aid in designing primer pairs. NCBI Primer design tool http://www.ncbi.nlm.nih.gov/tools/primer- blast/ and Primer 3http://frodo.wi.mit.edu/primer3/are recommended websites for this purpose.
l In order to avoid amplification of related pseudogenes or homologs it could be useful to run a blast on NCBI to check for the target specificity of the primers.
2. Prepare materials and reagents.
Arrange all reagents needed for the PCR experiment in a freshly filled ice bucket, and let them thaw completely before setting up a reaction.
1) Standard PCR reagents include a set of appropriate primers for the desired target gene or DNA segment to be amplified, DNA polymerase, a buffer for the specific DNA polymerase, deoxynucleotides (dNTPs), DNA template, and sterile water.
2) Additional reagents may include Magnesium salt Mg2+ (at a final concentration of 0.5 to 5.0 mM), Potassium salt K+ (at a final concentration of 35 to 100 mM), dimethylsulfoxide (DMSO; at a final concentration of 1- 10%), formamide (at a final concentration of 1.25-10%), bovine serum albumin (at a final concentration of 10-100 μg/ml), and Betaine (at a final concentration of 0.5 M to 2.5 M).
3. Setting up a Reaction Mixture
|
Template DNA (Plasmid remaining in PCR tube) |
≈1μl |
|
Forward Primer, 10 μM each |
0.5 μl |
|
Reverse Primer, 10 μM each |
0.5 μl |
|
2x PCR Mix |
10 μl |
|
ddH2O |
8 μl |
|
Total volume |
20μl |
4. PCR thermocycling.
|
Step |
Temperature |
Time |
|
Initial denaturation |
95°C |
2 min |
|
35 cycles |
95°C 60°C 72°C |
30 s 30 s 30 s / 1kb |
|
Final Extension |
72°C |
10 min |
|
Hold |
4°C |
- |
PCR product can be stored at -20°C for over one week.
Part 2: DNA electrophoresis in agarose gel
Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer ones. Thus, you can determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths).
A standard DNA agarose gel electrophoresis consists of following steps:
1. Preparation of the Gel
Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared using a w/v percentage solution. The concentration of agarose in a gel will depend on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The volume of the buffer should not be greater than 1/3 of the capacity of the flask.
Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA).
Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave, but can also be done over a Bunsen flame. At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved.
Add DNA staining reagent (Gel Blue) to the mixture. Alternatively, the gel may also be stained after electrophoresis in running buffer containing staining reagent for 15-30 min, followed by destaining in running buffer for an equal length of time.
Place the gel tray into the casting apparatus. Alternatively, one may also tape the open edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create the wells.
Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box before use.
2. Setting up of Gel Apparatus and Separation of DNA Fragments
Add loading dye to the DNA samples to be separated. Gel loading dye is typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel.
3. Observing Separated DNA fragments
Program the power supply to desired voltage (1-5V/cm between electrodes).
Replace the gel to the gel box. The cathode (black leads) should be closer the wells than the anode (red leads). Double check that the electrodes are plugged into the correct slots in the power supply.
Turn on the power. Run the gel until the dye has migrated to an appropriate distance.
Remove the gel from the gel tray and expose the gel to UV light. This is most commonly done using a gel documentation system. DNA bands should show up as fluorescent bands. Take a picture of the gel.
II. Procedure: PCR of target gene and gel electrophoresis verification
1. Design PCR primers for gene replication.
LacZα-F: 5’- ATGACCATGATTACGCCAA -3’ 19bp Tm = 53°C 42% GC LacZα-R: 5’- CTATGCGGCATCAGAGCA -3’ 18 bp Tm = 55°C 56% GC
2. Gene replication via PCR method
Prepare PCR replication system in the PCR microtube with plasmid remaining as follows:
|
Template DNA (Plasmid remaining in PCR tube) |
≈ 1μl |
|
Forward Primer, 10 μM each |
0.5 μl |
|
Reverse Primer, 10 μM each |
0.5 μl |
|
2x PCR Mix |
10 μl |
|
ddH2O |
8 μl |
|
Total volume |
20μl |
Transfer PCR tubes from ice to a PCR machine and begin thermocycling as follows:
|
Step |
Temperature |
Time |
|
Initial denaturation |
95。C |
2 min |
|
35 cycles |
95。C 60。C 72。C |
30 s 30 s 30 s / 1kb |
|
Final Extension |
72。C |
10 min |
|
Hold |
4。C |
- |
Note: Step 1 has been done by TA. Step 2 was done last week. Samples were stored at -20。C for one week.
3. Preparation of the Gel
Weigh out 0.6g agarose into an Erlenmeyer flask and add 60ml TAE buffer. Swirl to mix. Melt the agarose/buffer mixture by heating in a microwave. At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved.
4. Add 6µL DNA staining reagent (Gel Blue) to the mixture and mix well by gentle shaking. Place the gel tray into the casting apparatus. Place an appropriate comb into the gel mold to create the wells. Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature (about 20 minutes).
5. Add 2µL DNA 6x loading dye into the 10µL PCR system prepared last week. Mix well by gentle pipetting.
6. Remove the comb and place the gel in the gel box containing TAE buffer before use. Add 2.5µL Ladder and 10μl DNA samples into wells.
7. Replace the gel to the gel box. The cathode (black leads) should be closer the wells than the anode (red leads). Double check that the electrodes are plugged into the correct slots in the power supply.
8. Turn on the power (160V). Run the gel until the dye has migrated to an appropriate distance (About 15 minutes).
9. Gel imaging under UV exposure. DNA fragment analysis.
Experimental Datasheet of Exercise 5
Put here a picture of PCR sample gel electrophoresis result labeling your sample as well as DNA marker with different molecular weight. Try to analyze every band of your PCR sample.