Total Nucleic Acid Extraction

When you perform the conjugation lab, you should be able to observe the formation of a new strain of bacteria: E. coli HB101 cells that have acquired the pARO180 plasmid and thus acquired the ability to grow on ampicillin plates. When you see your plate results, you are observing the phenotype of the cells. In the next part of this lab, you can investigate whether you are able to see a DNA difference among the different strains correlating with their phenotypes.

Recall that there should now be three strains of E. coli present on various plates from your conjugation:

• E. coli S17 containing the plasmid pARO180. The plasmid has a gene providing resistance to ampicillin.
• E. coli HB101 with no plasmid. The chromosome contains a gene providing resistance to streptomycin.
• Conjugated E. coli HB101 with the plasmid pARO180. These cells are double-resistant, growing on plates that contain both amp and strep.

In addition, you should also have E. coli HB101 with the plasmid pGLO from that experiment. Now you can extract total nucleic acid from all four strains. Total nucleic acid means all the RNA and DNA from the cells. If you extract it and run it on a gel, you should see chromosomal DNA, plasmid DNA, and some RNA (mostly ribosomal RNA, along with messenger RNA and others). The chromosomes of all your strains should look the same on the gel; even though there are minor differences of nucleotide sequences, the chromosomes are the same size. However, some of the strains have the plasmid, and one doesn't. The challenge for this lab procedure is to see where the plasmid is present and where it's absent. The RNA is expected to cover a range of sizes and form a smear on the gel.

Gel results

Your gel for the strains in the conjugation lab might look something like this:

All three samples should contain chromosomal DNA. This acts as a positive control; if one of your samples has little or no chromosomal DNA, it's probably because you didn't get enough cells or didn't achieve good lysis (as described below).

Plasmid DNA is present in the amp-resistant cells (S17/pARO180) before conjugation and in the conjugated double-resistant cells (HB101 + pARO180), but not in the strep-resistant cells (HN101).

The RNA on the gel is mostly ribosomall RNA (rRNA), which is the most abundant RNA in the cell. There are two main fragments of rRNA, which are often visible as bands on the gel. In addition, there may also be a smear of smaller RNA molecules, which would include mRNA. With these methods,  the amount of RNA we recover is variable.

About the methods

We have used two different methods for total nucleic acid extraction in Bio 6B (SDS method and chloroform method). In both cases, the method has two main parts:

• Lyse (break open) the cells and release their contents into solution. The cell contents include DNA, RNA, proteins, and many other things. When you run the gel, you'll only see the DNA and RNA, because the gel is stained with ethidium bromide, which is specific for nucleic acids.
• Eliminate insoluble material by centrifugation. The insoluble materials (membranes, cell wall, some proteins) will form a solid pellet in your centrifuge tube. The DNA and RNA, which are highly water-soluble, will end up dissolved in the water.

The difference between our two methods is the way we perform lysis:

• SDS is a detergent. It dissolves membranes, causing lysis. It denatures and solubilizes many proteins and other cell components. This method is easy, because we don't need to use the fume hood. The SDS method makes for ugly, smeared gels, but recovers more DNA.
• Chloroform is an organic solvent. In this method, the cells are lysed by osmosis, when they are dispersed in pure water, along with the action of chloroform. This method seems to denature and precipitate many proteins. The chloroform method generally gives cleaner results on the gel, but often provides too little plasmid DNA to be visible. (This is not a standard method in molecular biology; we just tried it because we had some chlororm on hand, and we found that it works for us. A more common method is to use phenol + chloroform, which does a better job of cell lysis, but is more dangerous.)

Neither of these methods is optimized for providing pure DNA; for that, we will use plasmid purification in a later lab.

Procedure for total nucleic acid extraction: SDS method

Materials

• Plates from the conjugation lab (see the method below for which plates to use).
• Sterile toothpicks (These have been autoclaved and are in a closed container. Keep it closed, and don't take out a toothpick until you are ready to use it.)
• One large tube containing TE with 1% SDS. (TE, a dilute solution of Tris buffer and EDTA, is a common molecular biology solution. For this experiment, 1% SDS has been added to lyse the cells and solubilize the proteins.)
• 4 1.5-ml microcentrifuge tubes.

Method

1. You should have nine plates from the previous lab. Assuming that the previous lab worked as expected, you need these three conjugation plates for the next steps, along with one plate from the pGLO lab:
   • E. coli S17 containing the plasmid pARO180, growing on ampicillin. Label this strain "S" for the next step.
   • E. coli HB101 with no plasmid, growing on streptomycin. Label this strain "H."
   • Conjugated E. coli HB101 with the plasmid pARO180, growing on plates that contain both amp and strep. Label this strain "C."
   • E. coli HB101 with pGLO. This can be any of the pGLO transformation plates containing pGLO and ampicillin (plates 1, 2, or 3). Choose a plate with plenty of colonies; it doesn't matter whether the colonies are fluorescent. Remember that you will need to start pGLO cultures from your plates later, so don't scrape everything off the plate. Label this strain "pGLO."
2. Set your other six plates aside, or dispose of them in the biohazard waste.
3. Obtain four 1.5 ml microcentrifuge tubes. Pipet 300 μl of TE + SDS into each tube. Label them S, H, C, and pGLO, as listed above. Also put your initials on the tubes, because they need to go into the centrifuge along with other groups' tubes.

4. Use a toothpick to scrape some colonies from the S plate (E. coli S17 + pARO180). The picture below shows the amount of cells we tried to use in Winter 2018; this time, you should aim for about double this amount:

   

5. Disperse the cells into the S tube by twirling the toothpick. You should see the blob of cells come off the toothpick into the liquid. Close the tube.
6. Repeat the steps above to transfer cells from the H and C plates into their respective tubes, and for the pGLO plate.
7. Vortex the tubes briefly to disperse the blob of cells. The instructor will show you how to do this.
8. Incubate all the tubes at 70° C for 30 minutes.
9. Vortex the tubes for 10 seconds.
10. After incubation with SDS, the tubes will contain lysates with solubilized nucleic acid and protein, along with some insoluble material such as the cell wall. Centrifuge the tubes for 5 minutes at high speed to pellet the insoluble material.
11. Carefully pipet each supernatant into a new tube, leaving the pellet behind. There may be a blob of gelatinous material that makes your sample difficult to pipet. Try to get 100 μl of liquid without the blob; if necessary, you could pipet out the blob and then take the remaining liquid. If you're not running the gel today, you can stop at this point and put your tubes in the freezer until next time. Be sure they are properly labeled and put them in a rack (one rack for the whole lab section). (If you're running the gel today, you can take the supernatant from each tube, mix it with sample buffer, and load it directly on the gel.)

12. You started today with 9 conjugation plates. Save the three you used for nucleic acid extraction. Wrap a strip of stretchy vinyl tape (or parafilm) around the perimeter of each plate, sealing it up. Sealing your plates is a good idea when you're going to store them in the freezer, but a bad idea when you are culturing them in the incubator. Put your three sealed plates into a metal sleeve, along with other groups' plates; they will be stored in the refrigerator to keep the cells alive but growing slowly. If you haven't already tossed your other six plates in the biohazard waste, do so now.

    

Procedure for total nucleic acid extraction: Chloroform method

This is the method we used in Winter 2018; we won't use it in Spring 2018, but I'm leaving it here for reference.

Materials

• Plates from the conjugation lab
• Sterile toothpicks
• 4 tubes containing chloroform and TE (these tubes have already been prepared; the instructor will show you where they are).

Caution: chloroform is hazardous to touch or breathe and is very volatile (evaporates readily). Your instructor will tell you how to handle it. The tubes contain 100 μl of chloroform and 100 μl of TE, a dilute buffer dissolved in water. The aqueous phase (TE) sits on top of the organic phase (chloroform); the two don't mix. This is good, because it greatly reduces the amount of evaporation.

1. You should have nine plates from the previous lab. Assuming that the previous lab worked as expected, you need these three conjugation plates for the next steps, along with one plate from the pGLO lab:
   • E. coli S17 containing the plasmid pARO180, growing on ampicillin. Label this strain "S" for the next step.
   • E. coli HB101 with no plasmid, growing on streptomycin. Label this strain "H."
   • Conjugated E. coli HB101 with the plasmid pARO180, growing on plates that contain both amp and strep. Label this strain "C."
   • E. coli HB101 with pGLO. This can be any of the pGLO transformation plates containing pGLO and ampicillin (plates 1, 2, or 3). Choose a plate with plenty of colonies; it doesn't matter whether the colonies are fluorescent. Remember that you will need to start pGLO cultures from your plates later, so don't scrape everything off the plate.
2. Set your other six plates aside, or dispose of them in the biohazard waste.
3. Obtain 4 tubes of chloroform + TE. Label them S, H, and C, as listed above. Also put your initials on the tubes, because they need to go into the centrifuge along with other groups' tubes.

4. Use a toothpick to scrape some colonies from the S plate (E. coli S17 + pARO180). The amount of cells should look like this:

   

5. Disperse the cells into the S tube by twirling the toothpick. You should see the blob of cells come off the toothpick into the liquid. Close the tube.
6. Repeat the steps above to transfer cells from the H and C plates into their respective tubes, and for the pGLO.
7. Vortex the tubes for 30 seconds. The instructor will show you how to do this.
8. Centrifuge the tubes at high speed for 5 minutes. The small tubes must go inside a larger tube to fit in the centrifuge. Make sure the centrifuge is balanced. We may have an odd number of tubes; if so, one group can prepare one extra chloroform+TE+bacteria tube for balance.
9. After the spin, you should see two clearly defined layers. The chloroform (organic phase) is on the bottom; you don't want that. Most proteins have been denatured by the chloroform. The denatured proteins aggregate and precipitate, and often form a fuzzy or felt-like interface between the organic phase and the aqueous phase (TE). Be careful to avoid this interface. On top there is approximately 100 μl of aqueous phase; the nucleic acids, which are very hydrophilic, are in the aqueous phase. Carefully pipet 75 μl of the aqueous phase into a new tube. The exact amount isn't critical, but avoid the organic phase and the interface. Do this for all 3 tubes.
10. You should end up with 4 tubes of extracted nucleic acids (the aqueous phases). Be sure they are properly labeled and put them in a rack (one rack for the whole lab section). These are ready to be loaded onto a gel, and you'll do that in the next lab.
11. The tubes with chloroform are hazardous waste. The instructor will show you where to put them.

12. You started today with 9 plates. Save the three you used for nucleic acid extraction. Wrap a strip of stretchy vinyl tape (or parafilm) around the perimeter of each plate, sealing it up. Put your three sealed plates into a metal sleeve, along with other groups' plates; they will be stored in the refrigerator to keep the cells alive but growing slowly. If you haven't already tossed your other six plates in the biohazard waste, do so now.

    

Procedure for total nucleic acid gel

You'll run the gel the next lab period after you do the nucleic acid extraction.

Prepare the gel

Prepare your gel according to the instructions on the DNA electrophoresis page. Those instructions apply to all DNA gels.

For this total nucleic acid gel, prepare the gel with 0.8 % agarose.

Load the gel

If you used the SDS method and didn't centrifuge your tubes and transfer the supernatants, your tubes will contain lysates with solubilized nucleic acid and protein, along with some insoluble material such as the cell wall. Before running these samples on a gel, you should centrifuge them  to pellet the insoluble cell debris. Centrifuge the tubes at high speed for 5 minutes. The small tubes must go inside a larger tube to fit in the centrifuge. Make sure the centrifuge is balanced. If you have an odd number of tubes, prepare an extra tube with the same volume as your others.

Remember that each sample needs to be mixed with DNA loading buffer, as described on the electrophoresis page.

Follow the instructions in today's lab guide for gel loading.

Review

1. How could the total nucleic acid gel tell you whether conjugation occurred?
2. How will you recognize plasmid DNA?
3. Given a gel photo from this lab, you should be able to identify the bands on the gel.
4. Why do we use a gel with a relatively low agarose percentage for this experiment?