Information

Explanation of protocol in DNA extraction experiment

Explanation of protocol in DNA extraction experiment


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

In the Materials and Methods section of this paper on DNA extraction and analysis from hair I do not understand some parts of their protocol methods. In particular:

"The digested hair solution was then extracted twice with two volumes of phenol saturated by Tris-EDTA (TE) buffer (Sigma), and once with two volumes of chloroform. "

How do the volumes of phenol and chloroform come into play? I know they are used for an organic-solvent extraction, but what was the actual procedure used, i.e., the steps for adding each chemical?

Another question I had was on the buffer they used:

"0.01 M Tris-HCL(ph8), 0.005 M EDTA, 0.1 M NaC1, 0.039 M DTT, 2% SDS"

That whole combination would be the "Tris buffer" then yes? And the proteinase K would then be added to some of this buffer to digest the hair?


The DNA solution has a fixed volume (e.g., 0.1 mL). To a suitable test tube is added 0.2 mL of buffer-saturated phenol. After closing the test tube the mixture is mixed well, typically using a vortex mixer, but possibly by careful, repeated inversion. The aqueous phase and the organic phase are barely miscible. The tube is centrifuged to speed up the equilibration, and concentrate the denatured protein at the interface. After centrifugation the aqueous layer is carefully removed to a fresh test tube.

That is one organic extraction. This is repeated with a fresh aliquot of buffer-saturated phenol (another 0.2 mL). Then the aqueous phase is decanted again and extracted with 0.2 mL of CHCl3.

According to the recipe you provided, the buffer-saturated phenol was purchased from a vendor (Sigma-Aldrich Chemicals, of St. Louis, Missouri, USA), so you can look up the ingredients and composition on their online catalogue (no doubt).

The buffer recipe you provided is the proteinase K digestion buffer. It is typically performed at an elevated temperature.

This protocol is designed to isolate DNA from the nuclei of hair cells. Fully half of the contents of a eukaryotic nucleus are chromosomal proteins, by mass, so the proteinase K digestion step not only breaks open the hair cells, but also helps to remove all of the unwanted, contaminating cellular and nuclear proteins.


Explanation of protocol in DNA extraction experiment - Biology

  • piece of onion (approx 10 grams)
  • 2 large test tubes
  • mortar & pestle
  • graduated cylinder (10 or 50 ml)
  • 500 ml beaker, or mason jar
  • bamboo kabob skewer
  • nylon netting, or small dip net
  • funnel
  • small kitchen knife (or single-edge razor)
  • cold ethanol
  • ice bath (one per classroom may suffice)
  • Detergent solution= 1 part table salt + 1 part generic shampoo concentrate + 8 parts water
  • Enzyme solution= 1 part meat tenderizer powder + 19 parts water

Instructions : Write detailed notes on what you see and do at each step below.

1) Obtain a small piece of onion (about 10 cm 3 ) and chop it into tiny cubes (< 1 mm 3 )

2) Place the chopped onion into the mortar and thoroughly grind it with the pestle.

3) Add about 10 ml of detergent solution and grind again.

4) Filter the mixture through netting into a large test tube.

5) Add about 3-4 ml of enzyme solution to the test tube.

6) Let the mixture stand in a beaker of hot tap water for 10 minutes.

7) Place your test tube in the ice bath to chill for several minutes.

8) Carefully pour 10 ml ice-cold ethanol into test tube to form a separate layer on top. (You should see small wisps of gel forming at the boundary.)

9) Using the bamboo skewer, gently wind up the precipitated DNA. Show it to your instructor to verify that you have the DNA.

Why? The DNA is long and thin so it wraps around the toothpick like spaghetti on a fork!

10) One last question for you. Why didn't all that grinding and chopping destroy the DNA?

The DNA is so small and is coiled up so tightly by the histone proteins that it escapes most of the physical treatment. Once it is released from the histones by the enzyme solution, it unwinds into long strands and becomes much more fragile.


Protocol: DNA Extraction

I start nearly all of my experiments with the same procedure: extracting recombinant DNA from E. coli. In my research, I alter DNA sequences in order to create drugs that target specific types of cells in the human body. As a biological engineer, I stitch pieces of genes into circular pieces of DNA (plasmids) to create new cellular pathways. Though many of the protocols I use in the lab take a long time and have a high rate of failure, DNA extraction is simple, works 99% of the time, and takes less than 30 minutes.

Creating a new plasmid is an iterative process. As I add each new piece to my plasmid, I move the DNA in and out of E. coli cells. The cells act both as living storage and tiny Xerox machines, reproducing the DNA and filling the cells with copies that I’ll use in the next step of the process. I extract the DNA, cut and paste new genes into the plasmid, and insert it back into a fresh set of cells. Eventually I will harvest the complete plasmid from E. coli and transfer it into a yeast or animal cell.

Below is a general protocol for extracting plasmid DNA from E. coli bacteria cells. The overall goal is to separate the desired plasmid from other cellular components (RNA, protein, chromosomal DNA, etc.).

    I either start from a bacteria growing on a petri dish, or from vials of bacteria stored at -80 degrees Celsius. E. coli containing recombinant plasmids are typically stored frozen in glycerol, which serves as a cryoprotectant—just like frozen food, we don’t want our bacteria to get freezer burn. Using a sterile toothpick, I take a little bit of frozen bacteria and put it in a test tube containing Lysogeny Broth (LB often LB is referred to as Luria broth or Luria-Bertani broth, after the scientists who developed the medium). LB is a rich food for the bacteria that provides them with all the protein building blocks they need to grow.

Devin Burrill is a postdoctoral fellow at the Wyss Institute for Biologically Inspired Engineering at Harvard University. Her current research focuses on engineering targeted drugs for treating anemia, autoimmune disease, and viral infection.


Extracting DNA Using Magnetic Beads

Magnetic bead capture is the newest method of extracting DNA. Whole blood DNA isolation using magnetic beads works by capturing DNA on magnetic beads coated with a matrix of silica for binding nucleic acids.

As with the precipitation chemistry methods, the whole blood cells first must be lysed using SDS or similar detergents. The next step involves mixture of the lysed cells and magnetic beads, allowing DNA to bind the beads.

Several rounds of washing in the presence of a magnetic field then separates the DNA captured on magnetic beads from other unbound cellular contaminants. Then, a low-salt buffer elutes the DNA from the beads.

Some studies, including a review by Carpi et al., indicate that magnetic beads can be the fastest method. 1 However, this speed comes at a price—both monetarily and to your DNA yield and purity. 2 The magnetic bead approach can result in up to 20% reduction to your DNA yield. Also, any magnetic bead carryover contaminates your DNA sample and interferes with downstream applications. Lastly, it is the most expensive method because it requires magnetic capture stands, plates, and magnetic beads.


Teaching Challenge #1: How do I develop routines and procedures to support students to work independently in the science classroom?

Teaching Challenge #2: How can I develop a classroom culture that encourages student engagement, curiosity, and a desire to understand the world through scientific exploration?

These two interrelated challenges may not be entirely solved or conquered by this activity, however this activity represents several steps along the year-long journey (over which I get to accompany the student) and extending beyond my class. I hope that these experiences build toward a larger sense of independence and curiosity as they become adults.

Rather than starting from scratch, which would only lead to a number of dumbfounded looks and an awkward silence among the class, I direct students to use the DNA Extraction Lab Tempate and link provided. With this bit of scaffolding, students will study the protocol (per the online resource provided) and discuss the process in their teams. In other words, "what will it require for them to actually do this when they are green-lighted to go?"

In my mind I envision them as paratroopers waiting at the doorway of the C-17 cargo plane waiting for that "green light" that triggers the inevitable fight or flight response. I hope that today's prep will lead them to be eager to take the leap tomorrow rather than scurry as far away from the door as possible! This concept of a lab template is pretty standard in my classroom and can be explored a bit more from one of my previous lessons.

Next, students will agree to the specific steps that each member will perform for a fair and balanced team performance (on Day #2). They will then write up a protocol (based on the web link) that focuses on specific steps and the students in the team that will perform those steps.


Correlation to New York City Standards

S2a- The student demonstrates an understanding of the cell.

S4d- The student produces evidence that demonstrates understanding of the impact of technology such as constraints and trade-offs feedback benefits and risks and problems and solutions.

S5c- The student uses evidence from reliable sources to develop descriptions, explanations, and models.

S6a- The student uses technology and tools to observe and measure objects, organisms, and phenomena, directly, indirectly and remotely, with appropriate consideration of accuracy and precision.


DNA is most soluble in the water phase

So what does this have to do with the separation of DNA and protein?

Well in general, polar (charged) compounds dissolve best in polar solvents and non-polar molecules dissolve best in less polar or non-polar solvents.

DNA is a polar molecule due to the negative charges on it’s phosphate backbone, so it is very soluble in water and less so in phenol. This means that when the water(+DNA +protein) and phenol are mixed in the protocol, the DNA does not dissolve in the phenol, but remains in the water phase.


Results

Comparisons with Blood

Paired blood and fecal samples yielded identical genotypes for both replicates of all samples with the exception of two multiplexed loci (AE16 and MAF65) from one individual that gave consistent, but different genotypes for both replicates. Two additional replicate PCRs for both blood and fecal DNA all matched the results from the fecal DNA in the first runs, indicating that the DNA from blood produced false alleles in the first runs. One of those was from a well that showed notable dry-down. Both of these loci previously have shown tendencies occasionally to amplify false alleles that are alleles present in the populations studied.

Fecal DNA Extraction Experiments

Outer pellet material yielded consistently good results. Only when the outer pellet material was decreased to 15 mg did results begin to show a notable drop in PCR success ( Figure 1). For the three treatments that used 60mg of outer pellet material (two eliminated extra extraction steps), 8 of 12 samples yielded perfect PCR index scores of four and only 15 of 576 total amplifications were classified as less than good. Of those 15, 13 were reversed peaks and the remaining two were from one multiplexed reaction that was probably due to improper reaction conditions rather than amount of template DNA. Decreasing the amount of outer pellet material yielded a small, but measurable decrease in PCR success, especially at 15 mg, ( Figure 1 P = .022 for 60 mg versus 15 mg, P = .061 for 60 mg versus 30 mg ANOVAs of good amplifications).

Peak heights exhibited a somewhat different pattern of variation for treatments involving outer pellet material ( Figure 2) values for 30 mg and 15 mg both were notably lower compared with 60mg (P < .001), but not different from each other (P = 1.000).

There were obvious differences in PCR index values among the different pellet materials used when all treatments were compared ( Figure 1 P < .001 for all comparisons G tests for full extraction process) PCR amplification success varied inversely with the proportion of inner pellet material used. The use of inner pellet material had a similar effect on the peak height index ( Figure 2 P < .001 for all comparisons for full extraction process).

Different extraction procedures for outer pellet material yielded no significant differences for PCR outcomes (P = .177 ANOVA of number of good results). The peak height index similarly showed no effects from extending ethanol precipitation (P = 1.000), but exhibited a notable decline when bead-beating also was eliminated ( Figure 2 P = .013 relative to full process and 0.021 relative to no extension of ethanol precipitation). When samples were combined for each treatment involving whole pellet material, the pattern suggested improvements in PCR results from the additional extraction steps ( Figure 1), and the G-test for the three treatments was significant (P = .006). However, where any inner pellet material was included in DNA extractions, the four individual samples exhibited widely varying patterns for different treatments, including one sample (INY 1–15) for which the additional extraction steps substantially lowered the PCR success however, the highest PCR success for that sample included bead-beating ( Figure 3), indicating an interaction effect between bead-beating and extended alcohol precipitation. Because of the wide variation among samples, the ANOVA of number of good outcomes comparing extraction procedures for whole pellet material was not significant (P = .474). In contrast, peak heights produced significant differences for comparisons with the full extraction process for whole pellet material ( Figure 2 P = .012 for full process versus no extension of ethanol precipitation and P < .001 for full process versus no extension of ethanol precipitation or bead-beating) but the two altered extraction procedures were not different from each other (P = .151).

PCR index for treatments involving whole and inner pellet material by sample. Treatments for each sample from left to right are: whole pellet, full process whole pellet, no extension of ethanol precipitation whole pellet, no bead-beating or extended ethanol precipitation and inner pellet, full process.

PCR index for treatments involving whole and inner pellet material by sample. Treatments for each sample from left to right are: whole pellet, full process whole pellet, no extension of ethanol precipitation whole pellet, no bead-beating or extended ethanol precipitation and inner pellet, full process.

PCR results also showed considerable variation among samples where only inner pellet material was used ( Figure 3). Consequently, while the ANOVA of good PCR outcomes comparing the three different pellet materials was significant (P = .008), the only significant posthoc difference was outer pellet versus whole pellet (P = .008).

The low PCR index value for full extraction process of whole pellet material for sample INY 1–15 ( Figure 3) was identified in ANOVA of good results as a statistical outlier. Consequently, two additional replicate extractions from whole pellet material were carried out for this sample along with the same PCRs. Both replicate DNA extractions produced PCR index values close to the first (1.54, 1.56, and 1.62). This statistical outlier apparently represented important information rather than noise. Additionally, the closeness of the PCR index values among those three replicates suggests that this index is a sensitive and consistent measure of PCR success.

The extended alcohol precipitation incorporated in our full DNA extraction process was not only inconsistent in its effect on PCR outcome, it also increased the variance among samples in the peak height index for outer and whole pellet material ( Figure 2). For outer pellet material, this increased variance was due to a notable decline in the peak height index for a different sample (B76-8) than the one affected for whole pellet material. Overall, the peak height index exhibited an asymptotic relationship with the PCR index ( Figure 2).


Onion DNA Extraction Experiment

Preface
In this experiment, onions are used, this is because the onion has a low starch content, which allows the DNA to be clearly visible. Salt protects the negative end of the DNA phosphate, allowing the tip to come closer so that DNA can precipitate from a cold alcohol solution. Detergents cause cell membranes to break down by dissolving lipids and proteins from cells and disrupting bonds that hold the cell membranes together. Detergents then form complexes with lipids and proteins, causing them to precipitate the solution Onion (alium cepa)

Equipment and materials
Ethanol 95%.
Thermometer
Plate.
Measuring cup.
1000 mL bekaer glass.
Funnel.
Filter
Test tube
Freezer
Hot plate with bath
Ice Cube.
Spoon
Dishwashing soap or shampoo
Table salt, either iodized or not iodized
Distilled water
Great onions
Blender and knife to cut onion.
Timer or clock

3. Add the distilled water to the beaker to the final volume of 100 mL. Dissolve the salt by stirring slowly to avoid foaming.

4. Cut one large onion with a knife then blender and put in 1000 ml beaker glass


6. Input the 1000 mL glass of the DNA solution in a hot water bath at 55-60 ° C, as shown in the figure below, for 10-12 minutes.

8. Cooling the mixture in a water ice bath, about 4 ° C, as shown in the picture, below, for 5 minutes. In this process, press the onion DNA mixture against the side of the glass with the spoon back. This step slows DNA damage.



10. Remove the onion DNA solution into the reaction tube approximately 5 ml. Solutions can be stored in the refrigerator for about a day before use in the following steps.

11. Take a cold 95% ethanol solution from the freezer and add it to the test tube to make the ethanol layer above about 1 centimeter (cm). For best results, ethanol should be as cold as possible. Ethanol can be added to the solution

12. The DNA does not dissolve in ethanol. When ethanol is added to the mixture, all components of the mixture, except DNA, stay in the solution while the DNA settles out into the ethanol layer. The solution is allowed to stand for 2-3 minutes then white DNA will settle into the ethanol layer.



13. The formed DNA can be taken with a tooth or pipette or what stem of a crooked stirrer can take the DNA

The Classroom Flow: Lab Observations and Reflections

1. Students will settle into a quieter space as they observe their funnels and wait for their samples to be ready for extraction. There will be three basic phases:

  • The careful layering of the strawberry mixture and the alcohol
  • The insertion of the glass stirrer into the middle layer where the DNA is located
  • The slow extraction of the DNA out of the tube

2. Once students begin extracting their DNA, be on hand to encourage and congratulate! The kids love that type of feedback from their teachers especially during lab situations. It is a great opportunity to build community and connections as you share a common interest and collaborate to accomplish a task.

3. As students begin to clean up their materials and return to their lab tables to work on their analysis questions, remind students that they can be working on their lab observation/analysis questions as a team or looking up nucleotide definitions and diagrams in preparation for the last question on the lab document.


Watch the video: Protocol 1 - DNA Extraction Part 1 (October 2022).