Laboratory 11
DNA Part I (Extraction)
Introduction
Many different techniques have been developed for extracting DNA from bacterial cells
and environmental samples. Some methods are very complicated while others are quite simple.
Your choice of technique depends on the specific sample that you are working with and your
requirements for the quality of DNA extracted. All of the methods include: a step for breaking
open (lysing) the cells to release the DNA, a step for removing all of the proteins and other cell
components, and a step to precipitate the purified DNA. In this lab you will be using a relatively
simple method. The cells are lysed by exposing them to repeated freeze / thaw cycles and a
detergent (SDS). The proteins etc. are extracted away by using phenol:chloroform, and the DNA
is precipitated with salt and alcohol.
The DNA extracted in this lab will be used to assess the diversity of microbes in different
activated sludge samples.
Materials
Equipment
- micropipettes
- microcentrifuge
- Dry-ice / ethanol bath
- Hot water bath (37C)
- vortexer
Supplies
- microcentrifuge tubes
- micropipette tips
-
500 mM EDTA
lysis buffer
lysozyme solution
10% SDS
ice
phenol:chloroform
3.0 M sodium acetate
100% ethanol
sterile water
Procedures
Soil Extraction
1. Measure out 0.1 g of each soil sample into a microcentrifuge tube.
2. Suspend the soil in 75l of 500 mM EDTA by vortexing vigorously.
3. Freeze each sample for 30 seconds in dry-ice / ethanol bath
4. Thaw in a 37C water bath.
5. Repeat freeze-thaw cycle 2 more times.
6. Add 300 l of lysozyme solution in lysis buffer (~4mg/ml) and mix.
7. Incubate at 37˚C for 15 min.
8. Add 50 l of 10% SDS followed quickly by 800 l of phenol-chloroform.
9. Vortex for 1 minute to form an emulsion.
10. Spin in a micro-centrifuge at maximum speed (~14,000 RPM) for 3 minutes.
11. Remove the top phase with a pipette, avoiding the lower phase and any solids. Add this top
phase to a new microcentrifuge tube with 800 l of phenol-chloroform.
12. Vortex for 1 minute and spin for 3 minutes.
13. Remove the top layer and add it to a new microcentrifuge tube.
14. Add 50 l of 3.0 M sodium acetate and 1000 l of 100% ethanol and chill on ice.
15. Spin in microcentrifuge at max speed for 15 minutes.
16. Carefully remove the supernatant and allow the pellet to dry completely.
17. Re-suspend the pellet in 50 l of sterile water.
18. Freeze the pellet until the next period.
Laboratory 11
DNA Part II (Electrophoresis and PCR)
Introduction
The DNA extraction protocol that you used in part 1 of this lab should have resulted in
samples containing the mixed genomic DNA of all of the bacteria in the soil that you used. In
order to visualize the DNA, and to measure its size, we will be carrying out a simple gel
electrophoresis. Electrophoresis separates DNA according to its size by drawing it through an
agarose gel using an electric field. DNA is negatively charged so it is attracted to the positive
electrode in the chamber. As it moves through the agarose gel, larger pieces of DNA will be
slowed down more than smaller pieces. The absolute size of the DNA fragments is estimated by
comparing them to known standards.
After we have confirmed that DNA is present we will perform a PCR amplification to
isolate and detect the individual 16S rRNA genes from the mixed genomic DNA. The 16S
rRNA gene codes for a part of the ribosome and is present in all bacteria and archaea.
Differences in the DNA sequence of this gene can be used to distinguish between different
phylogenetic groups. PCR works by using short pieces of DNA (primers) that are homologous
to parts of the sequence of interest to locate a specific gene. Once the primers bind to the target
sequences, the stretch of DNA between them is copied. This process is repeated through many
cycles, and results in an exponential increase in the numbers of copies of the targeted gene.
Materials
Equipment
- thermal cycler
- gel electrophoresis power source
- electrophoresis rig
- micropipettes
- Polaroid camera
- UV light box
Supplies
- 1% agarose gel
- TAE buffer
- ethidium bromide(10 mg/ml)
- sterile water
- PCR kit
- Bacteria specific primers
- loading dye
- DNA size ladder
Procedures
Electrophoresis
1. Mix each sample with loading dye (2l dye with 10l sample) on a sheet of parafilm.
2. Place the 1% agarose gel into the electrophoresis box and cover with cold TAE buffer.
3. Load all 12l of each sample (including a size standard) into consecutive wells of the agarose
gel.
4. Place the cover on top of the electrophoresis box (check that the wires are attached to the
correct electrodes).
5. Set the power supply to run for 30 min. at 125V.
6. After the gel has run, remove it and transfer to the ethidium bromide-staining bath.
7. Stain for 10 min. then transfer the gel to the UV light box and take a picture.
PCR
1. Carefully label 1 PCR tube for each DNA sample, and 1 for a negative control (-).
2. Transfer 49l of the “master mix” into each tube.
3. Add 1l of each DNA sample to the appropriate PCR tubes and 1l of sterile water to the
negative control (-).
4. Load the samples into the thermal-cycler and start the program.
Standard protocol (for each 50l reaction)
40.75 l
1.0 l
1.0 l
1.0 l
5.0 l
0.25 l
1.0 l
PCR grade water
dNTP mix (10mM each)
5’ primer (~5 pmol/l)
3’ primer (~5 pmol/l)
10X NovaTaq buffer with MgCl2
(1.25 U) NovaTaq DNA polymerase
DNA template (~10 ng)
Temperature Cycles
Melting
Denaturing
Annealing
Extension
Final Extension
95C
94C
55C
72C
72C
5 min.
30 sec.
60 sec.
90 sec.
10 min.
TAE Buffer
4.84 g Tris Base
1.14 ml glacial acetic acid
2 ml 0.5 M EDTA
Agarose gel
1% agarose dissolved in 1X TAE
PCR Primers
Bacterial (bac) ~500 Bases
27f
5′ AGA GTT TGA TCC TGG CTC AG 3′
519r
5′ GWA TTA CCG CGG CKG CGT 3′
M = C or A; Y = C or T; K = G or T; R = A or G; S = G or C; W = A or T
Laboratory 11
DNA Part III (Electrophoresis)
Introduction
The product of PCR amplification is a large number of copies of the sequence targeted by
the primers used. When the genomic DNA that is used as a template for PCR is from a mixture
of bacterial species, then the PCR product will consist of many different, but similar, sequences.
The length of the products are determined by the position of the primers, therefore, the success of
PCR reactions can be evaluated by using gel electrophoresis to look for specific-length products.
In this lab we are using three sets of primers to to amplify parts of the 16S gene from
three phylogenetic groups that may be present in activated sludge. Two of the primer sets (bac
and arc) will result in products approximately 1350 bases long. The other (pla) will give a 633
base product. If the organisms were present in your sludge sample, then we will see a DNA band
of the appropriate size on the agarose gel.
Materials
Equipment
- gel electrophoresis power source
- electrophoresis rig
- micropipettes
- Polaroid camera
- UV light box
- 37˚C water bath
Supplies
- 1% agarose gels
- ethidium bromide
- loading buffer
- DNA size ladder
- Sterile distilled water
- Mnl-I reaction mix
- 0.75M sodium acetate
Procedures
Electrophoresis
1. Mix a sample from each PCR product with loading dye (2l dye with 10l sample).
2. Place the agarose gel into the electrophoresis box and cover with cold TAE buffer.
3. Load all 12l of each sample (including a size standard) into consecutive wells of the agarose
gel.
4. Place the cover on top of the electrophoresis box (check that the wires are attached to the
correct electrodes).
5. Set the power supply to run for 30 min. at 100V.
6. After the gel has run, remove it and transfer to the ethidium bromide-staining bath.
7. Stain for 10 min. then transfer the gel to the UV light box and take a picture.
Lab #11
Name:
20 points
Date:
Attach labeled copies of gel photos for DNA extraction and PCR products.