Colony PCR Protocol (modification of the current GLBRC Extension
Resources from Bioprospecting Lab)
Culture Unknown
Filamentous Fungal
Sample
Extract DNA:
Lysis Buffer
&
Thermocycler
PCR Amplification of
Fungal Universal ITS
region
Gel Electrophoresis
of PCR Product
Purification of PCR
product, Confirm on
Gel, and Prep for
Sequencing
Submission of PCR
Products for
Sequence Analysis
Bioinformatics:
Analysis of
Sequence by BLAST
This lab extension is intended for the identification of an unknown fungal species gathered from the
environment and analysis by DNA sequencing. Teachers and students that implement this extension activity
should have some background knowledge in molecular biology and have adequate laboratory skills.
Notes for Teacher:
This lesson is intended as an extension to the Great Lakes Bioenergy Research Center’s Bioprospecting Lab. Students will
have had an introduction to why cellulase enzymes in the environment are important in the future development of
cellulosic ethanol as a substitute for gasoline and hope for a low cost biofuel option that reduces dependence on foreign
oil, creates jobs, and reduces the impact of climate change. Students will use this information in context as they use
modern molecular techniques to identify the “mystery fungi” that is producing cellulase enzymes from their
environmental sample.
It would be recommend that the teacher lead the students through this lab and not provide a student handout. From
previous experience, this lab would be difficult for students to grasp and they will just ask you a lot of questions about
the procedure anyway. You may want to provide a simple powerpoint that may be helpful for you and the students to
improve the flow of this lab. A powerpoint is provided with this curriculum if you chose to use it; modify as needed.
Materials:
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Thermocycler
microcentrifuge
PCR tubes – two per sample to be tested
Micropipets and tips
Distilled/Deionized Water – 29.5 µL per sample
Lysis buffer – Protease K (20mg/mL – commercially available) – 1 µL per sample
Monoculture of isolate fungi species to be tested
PCR master Mix – includes dNTPs, Taq DNA Polymerase with buffers, and MgCl2 – 12.5 µL per sample
(Thermofisher, Promega PCR Master Mix, Cat # PR-M7501, $10.49/25 reactions)
Forward Primer – 0.5 µL per sample (~$10 from IDT,
http://www.idtdna.com/order/OrderEntry.aspx?type=dna&expand=10 )
Reverse Primer – 0.5 µL per sample (~$10 from IDT,
http://www.idtdna.com/order/OrderEntry.aspx?type=dna&expand=10 )
PCR product clean up kit – There are many companies that make PCR clean up kits.
o (Example: GeneJET Gel Extraction Kit, ThermoFisher, Cat # K0691;
http://www.thermoscientificbio.com/nucleic-acid-purification/genejet-gel-extraction-kit/ )
Access to materials for agarose gel electrophoresis & Transluminator
Funds for sequencing (MSU sequencing lab charges ~$7.50 per sample)
Additional Information on ITS Universal Fungal Primers:
Primers used for this lab are the Internal Transcribed Spacer (ITS) region universal primers for the detection and
identification of many fungal species. The primers for this lab were purchased from Intergrated DNA Technologies (see
attached sheet; website links are above). If you order the primers, you can order them online. The sequences of the
primers are as follows:
ITS1
5’- TCCGTAGGTGAACCTGCGG-3’
ITS4
5’- TCCTCCGCTTATTGATATGC-3’
For example, these primers will be shipped lyophilized (freeze-dried) and will need to be resuspended in sterile distilled
water as follows:
1.
2.
3.
4.
5.
6.
7.
Once you get the primers, look in the specification sheet for “amount of oligo”
It will be anywhere between 20 to 35 nMoles
Take that number, let’s say its 25 nMoles and add 25x20 µl of water so 500 µl total
If it’s 30 nMoles, add 30x20 µl of water so 600 µl total
If it’s 35 nMoles, add 35x20 µl of water so 700 µl total
The end concentration will be 50 picomoles = 50 pmol
We will use 0.5 µl of primers for every PCR reaction.
Procedure for Analysis:
1. Aliquot 19 µL of water and 1 µL of lysis buffer into each PCR tube. Lysis buffer, protease K (20 mg/mL),
will help to break up proteins and allow for better binding of the primers.
2. Using a micropipette tip from a P20 micropipet, scrap at the edge of a
pure fungal culture to obtain fungal hyphae into the micropipette tip.
You will only need ~ 1 µL of fungal biomass for this procedure to work.
Try and avoid getting agar into the tip. You may also be able to use a
sterile plastic toothpick to obtain your sample as well.
3. Mix the fungal biomass into a PCR tube containing water and lysis buffer.
4. Pipet the mixture up and down several times to mix the contents of the
tube. Make sure all the liquid stays at the bottom of the tube. You may
centrifuge the tube in a microcentrifuge to get all the contents to the bottom.
5. Place samples into a thermocycler for a cell lysis step. Lysis buffer works best at 50-65oC and 95-97oC
will kill anything remaining and help to lyse the cells and expose DNA for amplification. The
thermocycler settings are as follows:
 50oC for 30 min
 60oC for 20 min
 65oC for 10 min
 96oC for 10 min
6. (optional stop point) After lysis step in the thermocycler, samples can be placed in refrigerator until the
next day.
7. Pipet the contents of the PCR tubes up and down several times to mix.
8. Centrifuge at 2400 rpm for 2 minutes
9. PCR reaction must be prepared in new set of PCR tubes as follows:
 12.5 µL PCR Master Mix (contains DNA Taq Polymerase, MgCl2, and dNTPs)
 0.5 µL of each primer
 10.5 µL of water (it would be a good idea to autoclave water to remove DNase)
 1 µL of supernatant from DNA lysis mixture (tube that was centrifuged from above)
10. Samples are centrifuged briefly so as to bring entire contents of the tube to the bottom
11. Samples are placed in the thermocycler and the following PCR parameters are used:
 95oC for 5 min
 94oC for 1 min (Denature)
 58oC for 1 min (Annealing)
Run for 30 Cycles
o
 72 C for 2 min (Extend)
 72oC for 10 min
12. (optional stop point) Samples can be placed in the refrigerator overnight
13. The PCR products will need to be run on a 1% (w/v) agarose gel to verify PCR product. See next section
for details on how analyze PCR product.
Agarose Gel Electrophoresis Analysis:
1. Gel Electrophoresis can be prepared using the following conditions:
 1% (w/v) agarose in 1X TAE buffer
 Gel can be run at 100 Volts for 30 minutes using size and mass DNA marker (see below)
 Stained with Ethidium Bromide
2. Each lane received 5 µL of DNA. The lane with the marker received 5 µL at a concentration of 0.1µg
DNA per µL = 0.5 µg of DNA. Other lanes received 5 µL of PCR product.
3. Theoretically when running a gel, there should only be one (1) ~520 bp PCR product produced at the
ITS region (universal fungal). You will also have to estimate the amount of DNA you have in your PCR
tube by comparison with the DNA marker.
Example:


*5 µL added to each lane
The size of the PCR bands correspond to the correct length for our target sequence so the primers
amplified the correct sequence.
Since this is an estimate of mass, the intensity of the band from the 500 bp marker band is roughly
equivalent to the intensity of the PCR product. Therefore, based on the information from the standard
above and the picture of the gel, there is approximately 75 ng of DNA. There was 5 µL of PCR product
loaded onto the gel, therefore the concentration of DNA in the PCR reaction tube is approximately 15
ng/µL (75ng / 5 µL = 15 ng/µL).
Purifying the PCR Product
In order to continue on to DNA sequencing, you will need to clean up the DNA and separate it from the
unincorporated dNTPs, primers, etc. You will need to use a DNA purification kit to do this. We used the
ThermoFisher Gel Extraction kit to do this. This is a great and simple kit to use. Your students should not have
trouble performing this purification. We used the gel extraction kit because if you were to get multiple bands
of DNA you could actually cut the band of interest out of the gel and purify just the DNA sequence you are
interested in. In the example above, we only had one band (which is great) and so you can still use the gel
extraction kit, but we skipped the initial steps in the instruction manual and proceeded directly to step 6 in the
instructions. The instructions can be previewed from the website link provided above in the materials list.
In other words, the Gel Extraction Kit is great because it gives you the option to extract DNA directly from the
gel if you have multiple bands or to purify PCR product if you have a single band from your PCR reaction. In
the example above, we used DNA directly from the PCR tube and purified it rather than cut it out of the gel
because there is just one (1) single band.
Procedure for purification:
1. 15 µL from the original PCR tube was added to a new microcentrifuge tube.
2. 100 µL of wash buffer was added to the tube and mixed together by pipeting up and down.
3. The entire contents of the tube was loaded onto the surface of the purification media as part of the gel
extraction DNA purification kit.
4. At this point, I followed steps 6 – 10 of the gel extraction kit procedure.
Once you have purified your DNA, you must confirm that you actual have DNA to sequence at the end so you
will need to run the DNA on another 1% agarose gel to confirm your purification was successful. You will
perform the gel electrophoresis under the same conditions as described above. You will lose some DNA
during the purification process so you will need to make another estimation of DNA concentration in the tube
after purification.
5. When setting up your agarose gel, you will again use 5 µL of marker
6. Samples will be loaded using 2 µL of loading dye & 10 µL of purified PCR product
7. Gel will run for 30 minutes, stained, and analyzed
8. As before, since the PCR product’s band intensity is roughly equivalent to the 75ng 500bp standard
band, it can be extrapolated that there is approximately 75 ng of DNA PCR product on the gel.
9. Since there was 10 µL of PCR product DNA loaded onto the gel, there is approximately 7.5 ng/µL
(75ng/10µL = 7.5ng/µL) in the purified PCR product tube.
Preparation of DNA & Submission Procedure for Michigan State University Sequencing Lab
1. The information from the Michigan State University Sequencing Lab
(http://rtsf.natsci.msu.edu/genomics/sequencing-services/sanger/ ) recommends that DNA sequences be
prepared as follows before analysis:
DNA Type PCR Product
<100 – 200 bp
200 – 500 bp
500 – 1000 bp
1000 – 2000 bp
>2000 bp
DNA (ng)
Primer (pmoles)
2–6
6 - 15
10 - 40
20 - 80
80 - 200
30
30
30
30
30
Total Volume
12
12
12
12
12
Based on the information above, add
a) 2 µL of our PCR product (7.5 ng/µL)
b) 1 µL of ONE primer (ITS1 primer)
c) 9 µL of sterile water to a separate PCR tube, there will be enough DNA to run the sequencing
analysis.
Fill-out the MSU sequencing lab submission form as follows:
After 3-5 day, you will get an email verifying that your sequences are ready for analysis. You will be given a
username and password to view your DNA sequences at the website: http://finch.bch.msu.edu/Finch/ Click
on the “Folders” link to view your results.
Your sample submissions will be labeled as you provided them on the above submission form. In this
example, the samples submitted were labeled F1 – F4.
When you click on one of the submission links, you will see the following screen.
These options will
allow you to see
the actual DNA
chromatogram
Scroll down…..
This is your DNA
sequence from the
PCR product of your
unknown fungi!!
You can exclude the
crossed out
nucleotides in your
analysis.
Now that you have a DNA sequence to work with you can now move to analysis!
Identification of Unknown Fungal Specimen by DNA Sequence using BLAST
Once you receive the DNA sequence information from the lab, take the nucleotide sequence and use BLAST to
help you ID the organism.
1. Go to the URL: http://blast.ncbi.nlm.nih.gov/Blast.cgi and click on “nucleotide blast”
2. Place your DNA sequence into the box designated for sequence or “Enter Query Sequence”. Click on
“others” under database, and click on “somewhat similar sequences” under optimization. Finally, click
“BLAST”
3. The result from the sequence database query below indicates that this example sequence is strongly
associated with the organism, Fusarium chlamydorsporum. In this case, a quick Google search reveals
that this organism is a common soil fungus that is filamentous and associated with plants. The
organism appears over and over in the area near the top of the query. There may not always be a
perfect match between your unknown sequence and an organism found in the database. This is a tool
that takes time to learn how to use, but with practice, it is very powerful and can give you great
information about the identity of your unknown organism.
Happy bioprospecting! This activity can seem very overwhelming and difficult at first, but once you go
through the procedure a few times, it will surprise you how simple the whole process can be! This is a great
way for students to see how molecular biology can be used as a tool in environmental science. Good luck!
Need Help?
Toby West
Instructor
Lenawee Intermediate School District – TECH Center
Adrian, MI
(517) 263-2108
Toby.west@lisd.us
Acknowledgements:
This curriculum was created as part of the summer 2014 Research Experience for Teachers program through the Great Lakes Bioenergy Research
Center. The following individuals were critical in the development of this curriculum for classroom use.
Dr. Jonathan Walton
Professor of Plant Biology
612 Wilson Road, Room 210
Michigan State University
E. Lansing MI 48824
(517) 353-4885
fax: (517) 353-9168
walton@msu.edu
Dina Jabbour, PhD
DOE-GLBRC
Michigan State University
210 Plant Biology Building
612 Wilson Road
East Lansing, MI 48824
(517) 353-4886
Jeff Landgraf, PhD.
Michigan State University- RTSF
612 Wilson Rd., S-18 Plant Biology
East Lansing, MI 48824
Phone: 517-884-7302
E-mail: landgra1@msu.edu
http://genomics.msu.edu
Student Worksheet
Name: _____________________________________
Student Reflection: Colony PCR & Analysis
Over the past several lab periods, you went from collecting an environmental sample from the field that has
shown to have cellulase activity to sequencing that organisms DNA and identification using molecular biology
techniques. Now it is time to reflect on the process that you have just completed.
1. Using the key phrases below, put them in order as to how you completed this whole process. Some
boxes may be used more than once.
B. Agarose Gel
Electrophoresis
E. Analysis of DNA
sequence using BLAST
A. Isolated pure fungal
culture from the
environment.
D. Perform Polymerase
Chain Reaction (PCR) on
DNA unique to fungi
C. Purification of PCR
product
F. Extract DNA from
Fungi
Order of events:
_____
_____
_____
_____
_____
_____
_____
2. Why would it be essential to obtain a pure culture of fungi for this particular lab?
3. Which part of this whole process allowed for access to the fungus DNA?
4. In your own words, describe the process of PCR.
Use this link for a refresher on PCR
5. In order for a BLAST search of your unknown DNA to work, what information would be essential to
include in the BLAST database? Is it possible for your organism to NOT have a match in the database?
6. Do your results from your DNA sequence analysis make sense where you obtained your sample? In
other words, would it make sense if you obtained your sample from a soil sample in Michigan, but your
results say that your organism is only found in eastern Africa?
7. Given the information below, estimate the fragment length and approximate mass/µL (ng/µL) of the
DNA (lane 2) of interest if you loaded 10 µL of the DNA onto the gel?
DNA length: _________ bp
Estimated DNA Concentration: _________ ng/µL
70 ng
?
8. Given the following DNA sequence and using BLAST, what is the most likely identity of this organism? (You may
want your lab partner to read the sequence to you as you type it. Careful….don’t make a mutation!)
cgtgcgatac gatgaaagtt ggtggaaatc tggattccaa aggctatggt gtggcaaccc
61 ctaaaggctc agcattaagg tgggtggaat aatataacaa tatccgtgtt gttatagtat
121 tccacctacc ctgatgcatt ttgttgtcgt tttctttctt gtggattttg aggtaacttt
181 taaaagttta aaatctacaa tattccatgg agttaaataa gacggtaaat tatggtttca
241 tctatttaat gcatccattt tttttaatgt tctctctctc tgtgttgtcc tctctgactg
301 tttgttgctg ttttaatttt acagttcaac ggctttttca atttaaatgg taaaagccaa
361 gttatggtga cgaatgttaa ttgcatggat gtggtgttct tgttactttt tttctcaaag
421 acaatttccc catcccgcac acttcagttt tgagcaaatg ttatcctcca tgccaccttc
481 caatatttaa ccctgtattt gctgtacaga catttttata gctccacgtt ctgtgaaatt
541 tagccaattt gtcctcttgt gctccttttt ttatacgtta acgatttcct aagcatttgt
601 gcattttctt acaagttatg ttttatcgtt tcaagaaatg ctgttaacct ggcagtatta
661 aaactgaatg agcaaggcct cttggacaaa ttgaaa
What is the Genus species name? ________________________ Common name? ________________
9. What did you find most difficult about this whole process?
10. What did you like most about doing this lab?
Student Worksheet
Name: ___________KEY__________________________
Student Reflection: Colony PCR & Analysis
Over the past several lab periods, you went from collecting an environmental sample from the field that has
shown to have cellulase activity to sequencing that organisms DNA and identification of that organism using
molecular biology techniques. Now it is time to reflect on the process that you have just completed.
1. Using the key phrases below, put them in order as to how you completed this whole process. Some
boxes may be used more than once.
B. Agarose Gel
Electrophoresis
A. Isolated pure fungal
culture from the
environment.
E. Analysis of DNA
sequence using BLAST
D. Perform Polymerase
Chain Reaction (PCR) on
DNA unique to fungi
C. Purification of PCR
product
F. Extract DNA from
Fungi
Order of events:
___A_
__F__
__D__
__B__
__C__
__B__
__E__
2. Why would it be essential to obtain a pure culture of fungi for this particular lab?
If you are trying to identify ONE genus and ONE species then it is necessary to obtain a pure culture and not mix DNA
from multiple organisms. You will only want to amplify or “photocopy” DNA from one organism. Contamination could
be a real big problem with this lab.
3. Which part of this whole process allowed for access to the fungus DNA?
The use of protease K lysis buffer and heating step in the thermocycler broke up the cell walls and membranes enough
to allow for some DNA to spill out of the cells and be photocopies in PCR.
This QR code is optional.
4. In your own words, briefly describe the process of PCR.
You don’t need to use it if
you don’t want students
using cell phones in class.
PCR uses three repeating steps of 1) denaturing, which is where the double strands of DNA are separated. 2) Annealing,
which is where primers flanking are region of interest hybridize to the target DNA of the fungus. 3) Elongation or
extension, which is where the DNA Taq Polymerase binds to the primer and uses dNTPs to extend the complementary
strand of DNA thus making a “photocopy” of the DNA
5. In order for a BLAST search of your unknown DNA to work, what information would be essential to be
included in the BLAST database? Is it possible for your organism to NOT have a match in the database?
In order for you to successfully identify your organism, someone somewhere must have submitted a sequence the same
as yours or very similar and they knew the identity of the sequence they submitted to BLAST. It is possible that the
fungus that was sequenced may not be in the database and therefore could be a new species/subspecies!
6. Do your results from your DNA sequence analysis make sense where you obtained your sample? In
other words, would it make sense if you obtained your sample from a soil sample in Michigan, but your
results say that your organism is only found in eastern Africa?
Answers will vary, but students should make sure they do adequate self reflection and do basic analysis to insure that
their data/research makes sense.
7. Given the information below, estimate the fragment length and approximate mass/µL (ng/µL) of the
DNA (lane 2) of interest if you loaded 10µL of the DNA onto the gel?
DNA length: _~450_____ bp (answers may vary slightly)
70 ng
?
Estimated DNA Concentration: __7____ ng/uL (answers may
vary slightly)
8. Given the following DNA sequence and using BLAST, what is the most likely identity of this organism?
(You may want your lab partner to read the sequence to you as you type it. Careful….don’t make a
mutation!)
cgtgcgatac gatgaaagtt ggtggaaatc tggattccaa aggctatggt gtggcaaccc
61 ctaaaggctc agcattaagg tgggtggaat aatataacaa tatccgtgtt gttatagtat
121 tccacctacc ctgatgcatt ttgttgtcgt tttctttctt gtggattttg aggtaacttt
181 taaaagttta aaatctacaa tattccatgg agttaaataa gacggtaaat tatggtttca
241 tctatttaat gcatccattt tttttaatgt tctctctctc tgtgttgtcc tctctgactg
301 tttgttgctg ttttaatttt acagttcaac ggctttttca atttaaatgg taaaagccaa
361 gttatggtga cgaatgttaa ttgcatggat gtggtgttct tgttactttt tttctcaaag
421 acaatttccc catcccgcac acttcagttt tgagcaaatg ttatcctcca tgccaccttc
481 caatatttaa ccctgtattt gctgtacaga catttttata gctccacgtt ctgtgaaatt
541 tagccaattt gtcctcttgt gctccttttt ttatacgtta acgatttcct aagcatttgt
601 gcattttctt acaagttatg ttttatcgtt tcaagaaatg ctgttaacct ggcagtatta
661 aaactgaatg agcaaggcct cttggacaaa ttgaaa
What is the Genus species name? ___Mus musculus______ Common name? ___House Mouse______
9. What did you find most difficult about this whole process?
Answers will vary
10. What did you like the most about doing this lab?
Answers will vary
Connections to State and National Education Standards:
Next Generation Science Standards
HS-LS1-7: Use a model to illustrate that cellular respiration is a chemical process whereby the bonds
of food molecules and oxygen molecules are broken and the bonds in new compounds are formed
resulting in a net transfer of energy.
HS-LS-8: Evaluate the evidence for the role of group behavior on individual and species’ chances to
survive and reproduce.
HS-LS3-1: Ask questions to clarify relationships about the role of DNA and chromosomes in coding
the instructions for characteristic traits passed from parents to offspring.
HS-LS4-1: Communicate scientific information that common ancestry and biological evolution are
supported by multiple lines of empirical evidence.
HS-ETS1-1: Analyze a major global challenge to specify qualitative and quantitative criteria and
constraints for solutions that account for societal needs and wants.
Michigan High School Content Expectations (HSCEs): Biology
Standard B1: Inquiry, Reflection, and Social Implications
Scientific Inquiry
B1.1A Generate new questions that can be investigated in the laboratory or field.
B1.1B Evaluate the uncertainties or validity of scientific conclusions using an understanding of sources of
measurement error, the challenges of controlling variables, accuracy of data analysis, logic of argument,
logic of experimental design, and/or the dependence on underlying assumptions.
B1.1C Conduct scientific investigations using appropriate tools and techniques (e.g., selecting an
instrument that measures the desired quantity—length, volume, weight, time interval, temperature—with
the appropriate level of precision).
B1.1D Identify patterns in data and relate them to theoretical models.
B1.1E Describe a reason for a given conclusion using evidence from an investigation.
B1.1f Predict what would happen if the variables, methods, or timing of an investigation were changed.
B1.1g Use empirical evidence to explain and critique the reasoning used to draw a scientific conclusion or
explanation.
B1.1h Design and conduct a systematic scientific investigation that tests a hypothesis. Draw conclusions
from data presented in charts or tables.
B1.1i Distinguish between scientific explanations that are regarded as current scientific consensus and the
emerging questions that active researchers investigate.
Standard B2: Organization and Development of Living Systems
Maintaining Environmental Stability
B2.3A Describe how cells function in a narrow range of physical conditions, such as temperature and pH
(acidity), to perform life functions.
Cell Specialization
B2.4A Explain that living things can be classified based on structural, embryological, and molecular
(relatedness of DNA sequence) evidence.
B2.4d Analyze the relationships among organisms based on their shared physical, biochemical, genetic, and
cellular characteristics and functional processes.
Standard B3: Interdependence of Living Systems and the Environment
Environmental Factors
B3.5e Recognize that and describe how the physical or chemical environment may influence the rate,
extent, and nature of population dynamics within ecosystems.
Standard B4: Genetics
DNA
B4.2A Show that when mutations occur in sex cells, they can be passed on to offspring (inherited
mutations), but if they occur in other cells, they can be passed on to descendant cells only (noninherited
mutations).
B4.2B Recognize that every species has its own characteristic DNA sequence.
B4.2C Describe the structure and function of DNA.
B4.2D Predict the consequences that changes in the DNA composition of particular genes may have on an
organism (e.g., sickle cell anemia, other).
B4.2E Propose possible effects (on the genes) of exposing an organism to radiation and toxic chemicals.
Standard B5: Evolution and Biodiversity
Molecular Evidence
B5.2a Describe species as reproductively distinct groups of organisms that can be classified based on
morphological, behavioral, and molecular similarities.
B5.2b Explain that the degree of kinship between organisms or species can be estimated from the similarity
of their DNA and protein sequences.
Michigan High School Content Expectations (HSCEs): Chemistry
Standard C1: Inquiry, Reflection, and Social Implications
C1.1A Generate new questions that can be investigated in the laboratory or field.
C1.1B Evaluate the uncertainties or validity of scientific conclusions using an understanding of sources
of measurement error, the challenges of controlling variables, accuracy of data analysis, logic of
argument, logic of experimental design, and/or the dependence on underlying assumptions.
C1.1C Conduct scientific investigations using appropriate tools and techniques (e.g., selecting an
instrument that measures the desired quantity—length, volume, weight, time interval, temperature—
with the appropriate level of precision).
C1.1D Identify patterns in data and relate them to theoretical models.
C1.1E Describe a reason for a given conclusion using evidence from an investigation.
C1.1f Predict what would happen if the variables, methods, or timing of an investigation were changed.
C1.1g Based on empirical evidence, explain and critique the reasoning used to draw a scientific
conclusion or explanation
C1.1h Design and conduct a systematic scientific investigation that tests a hypothesis. Draw
conclusions from data presented in charts or tables.
C1.1i Distinguish between scientific explanations that are regarded as current scientific consensus and
the emerging questions that active researchers investigate.
Standard C5: Changes in Matter
C5.7 Acids and Bases
C5.7C Describe tests that can be used to distinguish an acid from a base.
Carbon Chemistry
C5.8C Recognize that proteins, starches, and other large biological molecules are polymers.