Laboratory Report

NAME ______Katheryne Avila Cortes____________

Lambda Restriction Enzyme: Perform restriction digestion of Lambda DNA and calculate the concentration of
the lactase gene to 20 ng/µL. Prepare the samples for loading into the Thermocycler for PCR gel analysis.
Lactase PCR: Prepare a 2% gel to resolve fragments at specific base pairs. Then, compare Lambda DNA on both
0.7% gel and 2% gel to determine the fragment size of each band to the ladder.
RNA Isolation: Isolate RNA from the cell pellet using solid-phase spin column method. Document
measurements using the Agilent TapeStation to determine RNA Integrity Number (RIN).
Tapestation: Load RNA samples into the Agilent TapeStation. This will provide a RIN quality score and detect
RNA markers, such as 28S and 18S rRNA bands. These bands will be assessed in a graph to evaluate their
intensities.

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 Procedure Summary

(1) Describe or show a picture of the calculations performed to normalize your patient DNA to 20ng/µL for
PCR (in order to load 100ng per reaction). If normalization was not necessary, show your calculations for
determining how many nanograms were instead loaded into each patient reaction.

(2) Insert the completed PCR master mix chart with all volumes shown. Show all calculations.

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(3) Consider some of the steps of the Invitrogen RNA isolation spin column procedure performed in lab.
Explain the purpose of the steps listed:
a. Add 350 µL of Lysis Buffer L3 to the tube of pelleted cells. Vortex to mix. Purpose:
Lysis buffer breaks open the cells and denatures proteins, releasing RNA into solution.

b. Add 350 µL of 70% ethanol and vortex. Pipette the lysate into the spin column and spin. Purpose:
RNA will bind to the silica membrane in the spin column as the ethanol will help adjust binding
conditions.

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 c. Add 350 µL of Wash Buffer W4 to the spin column and spin. Repeat. Purpose:
Wash buffer will remove residual salts, proteins, and contaminants while RNA remains bound to
the silica. After repeating, it will ensure maximum purity of the RNA before elution.

(4) Describe any deviations from or additions to the procedure given in the Lab Manual. Include any
additional calculations, reaction compositions, master mixes, etc. that were not listed above. If there were
no changes or deviations from the Lab Manual procedure, state N/A.
For RNA isolation, I added 350 µL of Lysis Buffer L3 to the Patient #212 tube and passed it along with 3 people to add
350 µL each. After that, we separately took 350 µL each to start adding 70% ethanol and continue with the procedure
for the RNA pellet.

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e

RESULTS

NOTE: Insert or describe all laboratory results here. Separate pages may be inserted from instrument documents (ie pdfs, jpeg) if
applicable, however, all results must be summarized or described by the student either on the document itself or in this results
section. Additional labeling may be necessary (identification of samples, controls, etc) .

You may type responses here or include pictures from lab notebook. Please crop pictures to include only the
requested information.
(1) Insert the jpeg of the 2% gel here (includes both lambda DNA reactions, ladders, and PCR results). Gel
must be labeled in some way (ex. Use annotation option on gel documentation system or label with text
boxes here in word. Handwritten annotations are acceptable, if the quality of photo is high).
Labels should include identity of each lane, resolved ladder bands must be properly labeled with molecular
weight. Other labels may be appropriate to identify important features of the results. Do not obscure the
bands on the gel.

(2) Insert cropped photos of your 0.7% (from Week 0) and 2% gels (from Week 3), showing just the cut
lambda DNA lanes. Place them side-by-side for comparison, 0.7% vs 2%. Label the bands of the lambda
DNA with their exact sizes (determined by your pre-lab), for both gels (only those bands which can be
resolved).
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 Lambda cut with BSTEII Lambda cut with EcoRI

(3) State the concentration and A260/280 ratio of the isolated RNA sample:
Concentration = 10.2 ng/µL
A260/280 ratio = 2.18
(4) State the RIN value for the RNA sample:
RIN value = 6.0

(5) Insert the results from the Agilent Tapestation for the RNA sample. Include the gel picture and the graph
showing the rRNA peaks. Include labels to indicate the identity of bands and peaks as needed.

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 My sample is on B1.

↑ smaller, low-
↑18S ↑28S
molecular weight
RNA

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 DISCUSSION/INTERPRETATION

NOTE: The results of the lab must be described in the student’s own words. Patient results must be clearly interpreted (genotype,
quantity, etc), if the lab includes a diagnosis. Results may need to be interpreted in the context of the disease being studied (ex.
affected by disease, carrier, prognosis of disease), if applicable. Validity of the test must be stated (control results, pass/fail) .

(1) Was the quality of the 2% gel acceptable? What are characteristics of a quality gel? Explain.
The quality of the 2% gel is acceptable because it showed sharp bands with minimal smearing in the wells.
There was no evidence of degradation. The characteristics of quality gel are clear, sharp bands with well-
formed wells that show even migration without abnormalities.
(2) Was the PCR experiment successful? How do you know? What does the positive control tell you?
- The PCR was successful due to the expected DNA band appearing at the correct size of the agarose gel.
The band was sharp, not smeared, and matched the predicted fragment size that indicated successful
amplification of the target DNA. There were minimal primer-dimers and the absence of non-specific
products.
- By comparing the PCR product lane to the ladder/marker can confirm the amplified fragment of the
correct size.
- The positive control tells me that it contains template DNA that is known to amplify with your primers.
Since the positive control shows the band, it proves that the PCR reagents and the thermocycler are
working properly.

(3) If you had included a negative template control (NTC) in this PCR experiment, what would be the expected
result?
The expected result should be no band in the NCT lane. Since there is no DNA template, the band would
indicate contamination of reagents or primer-dimer formation within the experimental sample.

(4) Observe the differences in the separation of Lambda DNA bands between your 0.7% gel and the 2% gel.
Describe how resolution is different between these gels.
The 0.7% gel shows larger pores and better resolution in large DNA fragments. The 2% gel shows smaller
pores and better resolution in small DNA fragments.

(5) Now compare the observed lambda DNA bands to the expected band sizes (from your pre-lab). Are all
bands accounted for? Which ones are missing or unresolved? Explain.
Not all bands were accounted for because there were some missing and unresolved. On a 0.7% gel, larger
fragments showed visibility, but smaller fragments were missing (224bp in BSTEII). However, on the 2%
gel, smaller bands were visible but larger fragments were faint (7,421 bp to 3,530bp in EcoRI). Showing
that lower percentage of gels are better for separating larger DNA while higher percentage of gels are
better for separating smaller fragments

(6) Discuss the results of the RNA isolation. Were all parameters high quality? If not, what could have caused
the observed deficiencies?

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 The RNA isolation parameters were not all high quality. My bands in 28S and 18S are present, but it is
lower than the ideal height. This could indicate partial degradation or loss of larger rRNA. The RIN of 6.0 is
below the recommended range so this could also mean RNase contamination, incomplete activation
during lysis, inadequate washing, or ethanol residue from the wash steps.
- To fix this problem: prevent RNase contamination by changing gloves often; work samples quickly;
make sure ethanol is removed before adding the wash buffer; and make sure to include the control
sample to confirm compliance.

TROUBLESHOOTING/CORRECTIVE ACTION

NOTE: Describe any troubleshooting that was performed during the experiment. Describe corrective actions taken, if needed. If all or
part of the lab experiment was repeated, describe the problem and the corrective action taken. Follow this with complete
documentation of the results and discussion of the repeated lab, in chronological order. Every repeated lab must be completely
documented, even unsuccessful attempts. All documentation (procedure modifications, results, interpretation) for a repeated lab
should be written in this section. Just tell the story of your lab!

(1) Was any troubleshooting necessary for this week’s lab? If not, state N/A. If so, state Yes and go to #2.
Yes
(2) Explain the troubleshooting that was performed and why you think problems occurred. Include any
repeated results (if applicable). Describe corrective actions (if applicable). If no corrective actions were
taken, describe what could have been done to improve the result.
During the RNA isolation, I had to add the Control sample to the TapeStation, but I forgot to add it. By
losing the reference, there is unknown comparison between RIN and fragment sizes across the run. What I
could have done differently is adding the control ladder and double check myself if I added the buffer with
the control. In addition, prepare another sample control to help spot the difference between the lanes.

This is what the control sample should look like in the TapeStation.

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 Save this document as a PDF prior to submission. Be sure to review for
appropriate presentation of information (labels in right place, etc). Be sure
inserted photos, pdfs, etc are high quality, adequate size, and readable.

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