LAB 8: Unknown Molecular Part II: Agarose Gel and PCR purification

Learning Objectives

  • Assess DNA quality and PCR results using agarose gel electrophoresis.
  • Purify DNA of chemical contaminants.
  • Determine the quantity and purity of DNA using spectrophotometry.

↓ Lab 8 Worksheet (.docx)

↓ Lab 8 Worksheet (.pdf)

Introduction

Last week, you extracted DNA from an unknown bacterial species. Then you set up a PCR to amplify the 16S rDNA gene. This week, you will check if the PCR was successful. If it was, you will prepare your sample for sequencing to determine the nucleotide sequence in the 16S rDNA gene. The resulting DNA sequence can be searched in a database to identify the unknown species.

Agarose Gel

We need to determine if your PCR of the 16S rDNA gene was successful. By running a small volume of your sample on an agarose gel, we can determine if any DNA was amplified in the PCR and if so, if it is the correct size.

On an agarose gel, smaller fragments of DNA travel faster through the gel than larger. By loading a sample consisting of standard sizes (called a molecular weight ladder), you can compare the size of products in your sample to known sizes of DNA. This will allow you to determine the size of your PCR products (Table 8.1). Depending on your primers, the DNA product will be 1465 nucleotides long (27F and 1492R primers) or 464 nucleotides long (341F and 805R primers).

Table 8.1. Most likely PCR and agarose gel results.
PCR Appearance on Gel Likely Reason

No amplification

No bands

Incorrect annealing temperature, reagent absent

Amplification of several DNA regions

Several bands

Annealing temperature too low

Amplification of target DNA only

One band of the correct size

Correct physical and chemical conditions met

You added genomic DNA to your PCR, but it will likely not be visible on the agarose gel. Genomic DNA is large (millions of nucleotides) and won’t travel very far on the gel.

The loading dye consists of three dyes. These dyes give you an indication of the location of DNA on the gel. If you run the gel for too long, your sample may run off the end of the gel and be lost in the buffer. Conversely, if you run the gel for too little time, your sample won’t separate well and it will be difficult to determine the size of any PCR products. By watching the location of the bromophenol blue band, you can ensure your sample is run the correct amount of time.

Table 8.2. Loading dyes and their approximate size in 1% agarose TAE gel.
Loading Dye Approximate Migration (size in nucleotides)

Xylene cyanol

4000

Bromophenol blue

300

Orange G

50

PCR Purification

Remaining in your PCR from last week, there are unused primers and DNA polymerase enzyme. These can interfere with the sequencing reaction. We need to remove everything except the DNA amplified by PCR.

To purify the DNA, your sample will be loaded onto a column. The column contains a silica resin that binds DNA under high salt conditions, while proteins (like the DNA polymerase enzyme) do not bind in these conditions. Then, the DNA is eluted (released) from the column under low salt conditions. The high and low salt conditions are created by passing different buffers (solutions of chemicals) through the column. The end result of PCR purification is 16S rDNA gene product in elution buffer or water.

DNA Quantification

The DNA sequencing process is simply another PCR. This time, it will be done using the 16S rDNA gene product as template with a primer that will bind to your PCR product. Because you have purified this PCR product, you may have a very high concentration of DNA. A PCR can be inhibited if there is too much template. You need to ensure this template DNA is in the optimal range for the sequencing reaction. To do this, you need to quantify the amount of DNA in your sample.

Double-stranded DNA absorbs at 260 nm. When this wavelength is passed through a sample with DNA, the amount of absorption corresponds to the amount of DNA present. The instrument is zeroed with the solution the DNA is suspended in, typically water or elution buffer.

After determining the result of DNA concentration, you will adjust it so that you have 5-20 ng/ul.

DNA Sequencing

This is performed by an organization outside the college. We are using the Sanger or dideoxy method of sequencing. Read about it here.

Agarose Gel

Materials

  • 50X TAE
  • Agarose powder
  • Loading buffer
  • Molecular weight ladder
  • PCR from last week
  • 6x loading buffer (contains dye)
  • Red Safe dye

Method

Only one gel needs to be made for every five groups.

  1. Make 200 ml of 1X TAE in distilled water, using the 50X stock.
    • Use the c1v1=c2v2 formula and solve for v1.
  2. Using 50 ml of 1X TAE, make a 1% agarose gel (w/v).
    • The agarose mass is calculated based on 1% of the volume of the gel. In other words, 1% of 50 ml is the mass in grams of agarose required.
  3. Add 2.5 μl RedSafe dye per 50 ml of gel solution.
    • We need to use this dye to see the DNA after running the gel.
  4. Microwave in short bursts of 20 seconds to melt the agarose into the buffer.
    • You should see no flecks of agarose in the solution.
  5. Handle the flask with hot hands and keep the opening pointed away from everyone in case it boils over.
  6. Cast the gel using the 6-well combs.
  7. Once the gel is solidified, remove the comb, and pour in the buffer.
  8. Load the gel: Mix 5 μl of sample with 1 μl of 6x loading dye on a piece of Parafilm.
    • Load the MW ladder in the middle lane.
    • Load 5 μl of your sample in another lane.
  9. Record the lane that you loaded your sample in.

Note: There are two types of dyes used in this gel.

  • RedSafe is for staining DNA to allow it to be seen under UV light. If you don’t add this, you won’t be able to see the DNA on the gel to know if your PCR was successful.
  • Loading dye is for marking where the DNA is on the gel while running. If you don’t add this, you won’t know when to stop running the gel, because you won’t know where the DNA is.
  1. Run the gel at 100 V approximately 45 minutes while you complete the next steps. Don’t let the orange dye run off the gel.
  2. Using the gel imager, take a picture of the gel. Determine the size of your PCR product by comparing to the MW ladder.
  3. Designate one person to handle the gel. Discard gloves immediately after.
  4. Dispose of the gel in marked, white pail.

PCR Purification

This is usually done only if you have one PCR product of the correct size, visualized on the agarose gel. We will complete these steps while the gel runs, so everyone can try this protocol.

Materials

  • Remaining PCR sample (approximately 45 µl)
  • QiaQuick Spin column
  • Buffers PB and PE
  • Nuclease-free water or buffer EB

Method

  1. Transfer the PCR product to a 2 ml tube. Label the tube with your initials.
  2. Add 5 volumes of Buffer PB to 1 volume of PCR sample and mix.
    • E.g. If the PCR volume is 50 µl, add 250 µl Buffer PB.
  3. Label a column on the side and top with your initials.
    • Handle the columns with great care, wearing clean gloves. Do not touch the base of the column to anything once removed from the bag. Place inside the collection tube immediately.
  4. Using a micropipette, apply the PCR sample to the column.
  5. Centrifuge the column at 13,000 rpm for 1 minute.
  6. Discard the flow-through into a small waste container.
    • Flow-through is the solution that is now in the collection tube.
  7. Add 750 µl Buffer PE to the column.
  8. Centrifuge at 13,000 rpm for 1 minute. Discard the flow through.
  9. Centrifuge the column at 13,000 for 1 minute to remove residual Buffer PE.
  10. Label a new microcentrifuge tube with your initials and unknown sample name.
  11. Place the column in the microcentrifuge tube.
  12. Place 50 ul Buffer EB directly onto the membrane, without touching the membrane with the tip (changing your tip between each sample). Incubate the column 1 minute at room temperature.
    • When you open the cap of the membrane and look down the column, you will see the white membrane. Aim for this as you drop on the EB.
  13. Centrifuge 1 minute at 13,000 rpm.
    • Record the location of each column in the centrifuge. Occasionally, the tops of the tubes snap off. If the location isn’t noted and both lids snap off, there is no way of knowing which sample is in which tube.
  14. Remove the column and store your tube on ice.

DNA Quantification

If you had DNA to purify, use the Qubit fluorimeter to quantify the DNA using the dsDNA high-specificity assay.

  • Select dsDNA as the assay on the touchscreen.
  • In a small tube, mix 2 µl PCR mix with 199 ul Qubit working solution. Incubate 2 minutes then read.
  • If this is too much, repeat with 1 µl PCR mix.
  • If this is too little, repeat with 10 µl PCR mix and 190 µl Qubit working solution.

Using c1v1=c2v2, you will adjust the DNA concentration of your sample using nuclease-free water.

Target C2 = 10 ng/μl

C1 = result from Qubit Fluorometer.

V1 = x µl

V2 = 10 µl

Amount of nuclease-free water to add:

10 – v1 = µl H2O to add

In a 1.5 ml tube, mix the volume of sample (v1) and nuclease free water.

341F will be used as the sequencing primer. The primer will be added at 2 µM concentration and 5 µl volume.

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