No LAB, Selected questions to aid in discussion.
1. Detail at least 2 reasons why your results turned out the way they did. This should be easy to do if your results are "unexpected", but even expected results can have multiple explanations.
-My results had low expression quality and I believe this may be because the tissue collected consisted of tube feet and tube feet are composed mainly of actin. -
-I saw a higher expression within the experimental groups compared to the control and this was expected because as the organisms were stressed I would expect resources to be diverted or halted increasing ion pump expression to combat the lowered salinity and predator proximity.
2. What are two obstacles that you encountered during your lab work and experimental design? Did these obstacles affect your results? Why?
-One problem with my experimental design was during the initial stages of video data, we could only see what was going on during the day when the lights were on. Since the urchins contain light receptive cells their behavior at night may be drastically different. This could definitely affect our results for covering behavior, the best thing to do is to look into previous research papers that may have examined behavior during the day and night cycles.
-Another obstacle we encountered was a lack of information as to where certain genes were more highly expressed in urchins. With my current results I would of looked into what was being expressed within urchin tube feet and changed what gene I am analyzing as a stress indicator. This may of affected my results because the quality of the expression was very poor.
3. Explain at least one aspect of your research and its results that have a greater impact outside of your own personal learning experience. What would you tell a non-scientist who challenged the importance of your research?
-The data shows how the green urchin responds to single and multiple stressors. My results suggest that ion pump expression was overall more highly expressed. However, when comparing the results from the predator only tank and the salinity/predator tank a pattern emerges that suggests the urchin does not respond as well with multiple stressors. Overall the experiment data suggests that stressors may act synergistically and degrade the urchin overall ability to respond to stress.
-When explaining this to a non-scientist I would explain how the experiment looked at only two stressors within a controlled environment and how the results suggests that each additional stressor degrades the organisms overall response or in this case ion pump regulation. The lab setting is nothing compared to the multitude of stressors organisms are experiencing within the wild population and it is a lack of understanding of how these events tie together that causes events such as mass mortalites and or reduction of important species. This research could have impacts on conservation, ecology, distribution, and even construction of buildings close to sea due to the impact or stress that it may have on the organism.
4. What part of your research and analysis has completely stumped you? Is there anything you can do to find the answer or will it always remain a mystery?
-I would like to understand more thoroughly why my group members qPCR quality was such a higher quality then my samples when we both used the same tissue and the same procedures to provide our results. This leads me to believe that it is not the tissue but the gene that I selected is poorly expressed within the tube feet of the green sea urchin. This could be solved by testing tissue at a higher concentration and then by comparing that with my current results. -
5. In about 3 sentences each, summarize 2 papers that you are going to cite in your own paper that give insight into the results that you found.
-Multiple factors explain the covering behavior in the green sea urchin, Strongylocentrotus droebachiensis by Cle'ment P. Dumont. This paper is very interesting because they explore both the day and night covering behavior observed in the green urchins. Also they look into other possibilities such as surge protection that might explain the covering behavior. Another unique point about this paper is that they compare the behavior of juveniles to adults, they found that juveniles express the covering behavior much more frequently then adult urchins.
-THE OSMOTIC ADJUSTMENT IN THE ECHINODERM, STRONGYLOCENTROTUS DROEBACHIENSIS by R. Lange. The paper describes the osmotic balance in the green sea urchin and the mechanisms involved in maintaining and isotonic equilibrium. I found this to be very interesting because it may explain why the quality of my qPCR was so poor. Initially I thought that the tube feet acting as an arm exposed to the external environment away from the protected body would work very well in ion movement.
22 November 2011
Summary:
-The purpose of Lab 8 was to run qPCR on all of the green urchin samples collected during the experiment.
Materials and Methods:
1. A master mix was made following the chart:
| Component |
Volume(uL) |
Multiplier |
TotalVolume(uL) |
|---|---|---|---|
| x2 Immomix |
12.5 |
x46 |
575 |
| Syto-13 dye (50uM) |
1 |
x46 |
46 |
| Up Stream Primer |
1.25 |
x46 |
57.5 |
| Down Stream Primer |
1.25 |
x46 |
57.5 |
| Di H2O |
7 |
x46 |
322 |
3. Placed x6 (8-well) well strips with labels 1-6 and my initials.
4. Master-mix was vortex-ed for 45 secs to ensure mixing.
5. 23ul of master mix was placed into each of the 46 wells.
6. The below chart corresponds to each cDNA sample in its respective well.
-2uL of cDNA was added to each respective well.
-2uL of DI H2O was added to Negative Controls.
| Strip Number |
Well#1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
|---|---|---|---|---|---|---|---|---|
| 1 |
Neg Control |
Neg Control |
C1 |
C1 |
C2 |
C2 |
C3 |
C3 |
| 2 |
C4 |
C4 |
C5 |
C5 |
C6 |
C6 |
P1 |
P1 |
| 3 |
P2 |
P2 |
P3 |
P3 |
P4 |
P4 |
P5 |
P5 |
| 4 |
P6 |
P6 |
P7 |
P7 |
S1 |
S1 |
S2 |
S2 |
| 5 |
S3 |
S3 |
S4 |
S4 |
S5 |
S5 |
S6 |
S6 |
| 6 |
S7 |
S7 |
Neg Control |
blank |
blank |
blank |
blank |
blank |
8. qPCR conditions:
1. 95°C for 10 minutes
2. 95°C for 15s
3. 55 °C for 15 s
4. 72°C for 30 s (+ plate read)
5. Return to step 2 39 more times
6. 95°C for 10s
Results:
The efficiency of gene expression was low (.76619) and for this calculation to be more accurate an average
efficiency of >.85 should be attained. The results for Na-K-2Cl co-transporter ion pumps varied considerably
with respect to magnitude as the three tests groups ranged from no expression to >1000x similar samples.
The control group was lower on average than both test groups with an average expression value of (5.12452E-09).
Predator only (2.74604E-06) and predator with salinity (2.45701E-06) were markedly higher on average. Three samples
one from each group showed no expression (S1, P3, C6). Two samples, one from predator only (P2 - 1.92E-05) and the
other from Salinity + Predator (S2 - 1.71E-05) showed concentrations >1000x all other expression values.
Conclusion:
While the data suggests that increased stress levels lowers the urchins ability to maintain ion pumps there is the issue
with degraded or inadequate protein expression form the tissue samples. Judging by the difference between the quality
of my RNA expression and Miranda's it is not an issue of tissue collection. Rather it is an issue with gene selection,
possibly the tube feet which contain mostly actin lacked a significant quantity of ion pumps within the tissue.
Next Step:
-analysis data and write research paper.
Reflection:
-I really liked this lab as I am one step closer to seeing the results of our urchin experiment!
15 November 2011
Summary:
-The purpose of the lab 7 was to:
1. Perform dot blot procedures on c.gigas DNA.
2. Run PCR on urchin samples to verify primers.
3. Run qPCR procedures on urchin sample.
Materials and Methods:
Dot Blot Procedures - Dilution
1. Obtained human DNA at a concentration of 302ng/ul
2. Made a 50ng/nl dilution of sample.
C1V1 = C2V2
(302)(V1) = (50)(40)
V1 = 6.6ul of DNA used for dilution
33.4ul of DI H2O and 6.6ul of human DNA.
3. Used dilution to create 5 additional dilutions using the table found in Lab 7.
| Dilution |
TARGET amount |
ul of H20 |
ul of 20X SSC |
ul of 50ng/ul DNA sample |
| 1 |
800 ng |
124 |
60 |
16 |
| 2 |
400 ng |
132 |
60 |
8 |
| 3 |
200 ng |
136 |
60 |
4 |
| 4 |
100 ng |
138 |
60 |
2 |
| 5 |
50 ng |
139 |
60 |
1 |
1. Nylon membrane soaked in X6 concentrated SSC, for 10 minutes.
2. DNA samples allowed to denature in boiling water for 10 minutes.
3. Applied 500ul of SCC to each well and allowed to filter through with vacuum. Adjust vacuum so that all liquid takes approximately 2 minutes to sink through filter paper.
4. Transfer entire DNA sample to appropriate wells.
5. Filter samples through membrane.
7. Transferred membrane dot side up to treated filter paper, Filter paper treated with de-naturation (10min) and neutralization buffer (5min).
8. Transferred membrane to dry on dry filter paper.
9. Transfer dot blot to UV trans-luminator for 2 minutes, DNA side down.
WesternBreeze® CHROMOGENIC IMMUNODETECTION
1. Prepare a 20ml solution containing -
- a. Ultra filtered Water 14 ml
- b. Blocker/Diluent (Part A) 4 ml
- c. Blocker/Diluent (Part B) 2 ml
- d. Total Volume 20 ml
2. Cover membrane in plastic dish with 10ml of blocking solution.
3. Incubate for 30min on rotary shaker at 1 rev/min
4. Then decant the blocking solution.
5. Rinse for 5 mins with 20 ml of water, then decant and repeat.
6. Make 10 mL of Primary Antibody Solution containing -
- a. Blocking Solution 10 ml
- b. 5-MeC antibody 2 µl
- c. Total Volume 10 ml
7. Incubate with solution 10ml for 1 hour.
8. Wash membrane for 5 mins and repeat 3 times.
9. Rinse with 20ml of water for 5mins, repeat x2.
10. Incubate in 5ml of Chromogenic Substrate until color appears.
11. Rinse with 20ml of water for 2 mins.
12. Dry with clean piece of filter paper.
PCR:
1. PCR was done to Labs1-3 Protocol which can be found here:
Lab 1 Lab 2 Lab 3
2. Samples ran - C1, P1, S1
qPCR Procedures:
1. Prepare for number of reactions and +1 for error.
2. Use mixing chart.
3. Add master mix to well plate.
4. Add 2ul of cDNA to samples.
5. Add 2ul of di H2O to negative controls.
6. Cap wells.
7. Load plate:
qPCR conditions:
1. 95°C for 10 minutes
2. 95°C for 15s
3. 55 °C for 15 s
4. 72°C for 30 s (+ plate read)
5. Return to step 2 39 more times
6. 95°C for 10s
Conclusions:
1. Dot blot showed positive results for DNA methylation.
2. PCR proved primers are good and working, and that RNA is not of great quality.
3. qPCR results showed that my primers may be amplifying multiple items, could be primer dimer
-Also my samples dont appear to begin amplification until 30+ cycles.
Reflection:
-I need to run collective qPCR for all of my samples to understand what the effects of predators and salinity change had on the ion pumps of green sea urchins.
-I understand the dot blot technique better and what the results represent.
10 November 2011 (Thursday)
Summary:
-Completed reverse transcription procedures.
Materials and Methods:
Reverse Transcription-
Reverse Transcription Procedures:
1. Added 2ug of TOTAL RNA to appropriate PCR tube.
2. Added 1ul of Oglio DT, no H2O
3. Incubated for 5min and prepared master mix.
| Name |
Amount(uL) |
| M-MLV 5X Reaction Buffer |
5 * 21 = 105ul |
| dNTPs |
5* 21 = 105ul |
| M-MLV RT |
21ul |
x1- 90C for 5 min,x40 cycles of (90C for 15s, 55C for 15s, 72C for 15s), x1- 71 for 5 mins.
Conclusion:
-Will run gel on Tuesday 15th November.
Reflection:
-Accidently left unused RNA samples in ice bucket overnight, most likely they are no longer good and will need to be thrown away.
8 November 2011
Summary:
-Completed RNA extraction procedures for 20 Green Urchin Predator Response experiment (GUPR).
Materials and Methods:
-RNA extraction part 1:
1. Labeling is as follows (C1-6) = Controls, (P1-7) = Predator Only, (S1-7) = Salinity Dec. and Predator.
2. Added 500ul Tri-reagent and homogenized.
3. Once homogenized we added 500ul of additional Tri-reagent, then vortexed for 15secs.
(We did not turn on the heating block as it was not required to assist in separation.)
4. Incubated for 5-6 mins and then added 200ul Chloroform under the fume hood.
5. Vortex for 30s.
6. Incubated for 15s and then placed into 4C centrifuge at maximum speed for 15min.
(8 of 20 samples did not completely separate and had to be reran through the vortex and then centrifuge again.)
7. Transferred the aqueous phase to new 1.5ml tube. Disposed rest of material in waste jar. (Do all this under fume hood!)
8. Added 500ul isopropanol. to each sample and inverted several times.
9. Incubated for 10 mins then spun down at max speed for 8 mins.
10. Removed supernatant.
11. Added 1ml of 75% ETOH and spun at 7500 rpm for 5 mins.
12. Removed EtOH and dried inside tubes with chemwipe, did not touch RNA pellet.
13. Suspended the pellet in 100uL of 0.1%DEPC-H2O.
14. Ran Nano-Drop on all 20 samples.
Results of Nano-Drop:
| Sample |
ng/ul |
Amount Needed for1ug of RNA |
x2ug per Reaction |
| C1 |
40.6 |
24.6 |
49.2 |
| C2 |
31.2 |
32.1 |
64.2 |
| C3 |
9.3 |
107.5 |
215 |
| C4 |
22.5 |
44.4 |
88.8 |
| C5 |
17.0 |
58.8 |
117.6 |
| C6 |
22.5 |
44.4 |
88.8 |
| P1 |
61.5 |
16.3 |
32.6 |
| P2 |
17 |
58.8 |
117.6 |
| P3 |
28.2 |
35.5 |
71 |
| P4 |
53.2 |
x |
37.6 |
| P5 |
21.3 |
x |
93.9 |
| P6 |
28.1 |
x |
71.2 |
| P7 |
47.7 |
x |
41.9 |
| S1 |
27.6 |
x |
72.5 |
| S2 |
60.9 |
x |
32.8 |
| S3 |
25.8 |
x |
77.5 |
| S4 |
55.4 |
x |
36.1 |
| S5 |
47.0 |
x |
42.6 |
| S6 |
47.2 |
x |
42.4 |
| S7 |
35.5 |
x |
56.3 |
-Wont know the results till we run the gel.
1 November 2011
Summary:
-Ran cDNA samples IGF1 and IGF2 on gel.
-Reviewed electrophoresis and western blot procedures.
-Prepared urchin gonadal tissue for protein extraction.
Methods and Materials:
-Electrophoresis Procedures
1. Decide on what percent of agarose you want (up to 2%).
2. Find the total volume of the mixture and use your intended percentage of aragose.
3. Weigh agarose 2g and mix it with 150ml TAE buffer
4. Microwave solution for 3 min, be careful its very hot.
5. Allow to cool for about 20min and then add 12ul of EtBr.
6. Pour into mold and let the solution solidify, when hardened wrap in plastic and place in -20C fridge.
-Running the Gel
1. Place gel on electrophoresis plate and fill with TAE until it is submerged.
2. Place known ladder 5ul in well of choice, usually far left or right.
3. Place samples 25ul each into each well.
4. Connect correct negative and positive terminals, remember "Run to RED"!
5. Turn on voltage to 100 volts for 20-30 min.
6. When finished, remove power, and place gel under UV light to examine amplified product.
-Western Blot
-Transfer
1. Soak filter paper in Tris-Glycine Transfer Buffer for 15 minutes.
2. Make a "blotting sandwhich"
- Anode (+++)
- filter paper
- membrane
- gel
- filter paper
- cathode (---)
-For further explanation click here!
Conclusion:
-Results from gel show a amplification in 300, 700 range and negative controls.
-IGF primers need to be reworked, not usable as is.
-Ready to begin RNA isolation of Urchin tube feet.
Gel Analysis:
-Our group was looking into IGF (Insulin Growth Factors) on C. gigas. Our conclusion is that the primer amplified multiple
genes at 300, 700 bps resulting in an inefficient primer. Our controls were clean.
-Click here for sample layout ROW 1 -------ROW 2 and hyperladder.
-Copy of Electrophoresis Gel can be found here. ROW 1 -------- ROW 2
Western Blot Analysis:
- Only the Urchin group showed positive for HSP 70 protein.
-Copy of the SDS gel can be found here.
-Copy of the sample list can be found here.
Reflection:
-Need to review western blot procedures as they were covered to quickly.
-Excited to start sea urchin co-transporter ion RNA isolation next week.
25 October 2011
Summary:
-Reviewed PCR procedures.
-Performed PCR on samples to protocol.
-Obtained tissue samples from green sea urchin.
-Dissected urchin from each group for protein extraction.
Methods and Materials:
-PCR
1. Made master mix for x5 reactions, 5th reaction was for pipetting error.
| Reagent |
1.reaction |
x.reactions |
Totals uL |
| 5x GoTaq Green buffer |
10 µL |
5 |
50 |
| 10mM dNTP mix |
1 µL |
5 |
5 |
| 10 µM forward primer |
1 µL |
5 |
5 |
| 10 µM reverse primer |
1 µL |
5 |
5 |
| GoTaq polymerase |
0.25 µL |
5 |
1.25 |
| nuclease-free water |
36.75 |
5 |
183.75 |
| Totals |
50uL |
n/a |
250uL |
| Tube |
2uL |
| Oyster |
cDNA |
| Oyster |
cDNA |
| Control |
H2O |
| Control |
H2O |
| Temp(C) |
Time |
| 92 |
5Mins |
| 92 |
15s |
| 55 |
15s |
| 72 |
15s |
| 72 |
5Mins |
-Removed tube feet from each specimen by placing an urchin in several inches of water and then snipping and collecting tissue.
-Collected about 10-15 tube feet per urchin.
-Dissected one urchin from each group for protein extraction. The urchin was dissected laterally around its equator and then the top was pulled back where we collected its digestive track and secondly gonadal tissue.
-All samples were immediately placed on dry ice and then moved to the -80C freezer.
Conclusion:
-Need to begin analysis of urchin tissue utilizing ion pump primers.
Reflection:
-Also should look into Hsp70 protein expression in addition to ion pump expression as hsp's are used within organisms as a general response mechanism.
18 October 2011
Summary:
-Performed reverse transcription procedures
-Developed experimental design and finalized
-Began experimental set-up
Methods and Materials:
-Reverse Transcription
1. Mix your stock RNA by pipetting or inverting.
2. Label new tube and add 5ul of your total RNA to tube
3. Add 1ul oligo DT and 4ul di H2O
4. Incubate the mixture for five min at 70C and then transfer to ice.
5. Add 5ul of M-MLV 5X reaction buffer.
6. Add 5ul dNTPs
7. Add 1ul of M-MLV RT and 4ul of di H2O
8. Incubate for 60 min at 42C and then heat inactivate at 70C for 3 min.
9. Spin down the sample
10. Store at -20C
-Experimental setup can be found here.
Conclusion:
-Stored sample in -80C freezer
Next Step:
-Run primers and PCR
-Begin experiment Thursday 20th OCT 2011
Reflections:
-I am ready to begin our experiment with the sea urchins as I have wanted an answer to this question for some time.
11 October 2011
Summary:
-Reviewed second of RNA extraction procedure.
-Performed remaining RNA extraction procedures.
-Ran quantitative analysis on RNA sample using Nanodrop spectrophotometer.
-Stored samples.
Methods and Materials:
-RNA Extraction Part 2:
(note): Turn on heat bath to 55C for later in the procedure.
1. Incubate for five mins at RT.
2. Add 200ul of chloroform to sample, under hood.
3. Now vortex at hgih speed for at least 30 seconds, should look milky.
4. Incubate for five mins RT.
5. Spin in refrigerated centrifuge for 15mins at max speed.
(note): Helpful tip when centrifuging samples place the "tip" end of the cap facing up.
6. Transfer aqueous phase ONLY (clear part) to fresh tube.
7. Dispose the inter-phase properly as it contains harmful chemicals.
8. Add 500ul of isopropanol to RNA.
9. Mix and incubate for 10min at RT.
10. Spin for 8mins at max speed.
11. Remove supernatant, be sure not to remove pellet!
12. add 1ml of 75% EtOH and allow to dry for no longer then 5 mins at RT.
13. Spin at 7500g for 5mins.
14. Remove supernatant, do not remove pellet.
15. Suspend pellt by adding 0.1% DEPC water and mix y pipetting.
16. Incubate for 5 mins at 55C
17. Remove flick several times and store on ice.
-RNA Quantification:
Note: Keep samples on ice as much as possible!
1. Pipette 2ul of 0.1% DEPC water onto the nanodrop.
Note: The blank sample of DEPC water allows the machine to recognize the "background" and
essentially ignore it when you run your sample.
2. Run Blank
3. Pipette 2ul of Your RNA sample onto pedestal.
4. Click Measure.
5. Follow protocol for interpreting your results but essentially you want A260/280 ratio of 1.82 or close.
6. Store sample at -80C.
Recordings and Measurements:
-Quantification results:
| Concentration |
292.3ng/ul |
| Ratio A260/280 |
1.85 |
| A260/230 |
1.94 |
-RNA extraction results showed a good ratio of 1.85.
Next Step:
-Start cDNA procedure on RNA sample.
-Create group projects.
Reflections:
-To understand the finishing steps of RNA extraction and then learn to operate the Nanodrop spectrophotometer.
05 October 2011
Summary:
-Reviewed basic molecular terms and functions.
-Began RNA isolation procedures on C. gigas gill tissue to protocol, stopped procedure after homogenization step.
-Ran protein isolation procedure on C. gigas digestive gland tissue using CelLytic MT and Bradford Assay.
-Learned how to operate the spectrophotometer.
Methods and Materials:
-RNA Isolation:
1. Mark snap cap with name, date, and tissue type.
2. Add tissue and then add 500ul of Tri-Reagent.
3. Homogenize sample with pestle.
4. Add another 500ul of Tri-Reagent.
5. Then vortex sample for 15secs.
6. Store on ice.
-Protein Extraction:
1. Record weight and tissue type, then label tube.
2. Add 500ul of CelLytic MT to the sample.
3. Homogenize sample
4. Invert tube several times, then place in refrigerated centrifuge at max speed for 10min.
5. Label new tube with "protein", add initials, organism, date.
6. Transfer the supernatant to the fresh tube.
-Protein Quantification:
1. Label new tube with "protein" and add BA for Bradford Assay with initials and date.
2. Dilute 15ul protein sample with DI water at a ratio of 1:2.
3. Label second tube "blank" and transfer 30ul of DI water to it.
4. Mix tubes by pipetting to ensure mixing.
5. To both tubes add 1.5ml of BA and invert to mix then incubate at room temperature for 10mins.
6. First set up spectrophotometer with blank ensure the absorbance reads zero. at what wavelength? -
7. Secondly place in protein assay and measure absorbance, record this value.
8. Mix by pipetting then repeat measuring procedure, record this value.
9. Take the average of the two values and plug it into the line equation (below), remember to account for the dilution.
10. Record concentration of protein.
Recordings and Measurments:
1. RNA Isolation procedure used 0.02g C. gigas gill tissue.
-Added a total of 1000ul of Tri-Reagent
2. Protein Extraction procedure used 0.053g of digestive gland tissue.
| Sample |
Reading |
| First absorbance |
0.261 |
| Second absorbance |
0.262 |
| Average |
0.2615 |
-For protein extraction: Concentration - 530.27ug/ml
Calculation - y=mx+b
m=1013.9
x=0.2615 * 2 (to account for the dilution)
b=0
y=1013.9(2*0.2615)
y=530.27ug/ml
Conclusion:
-RNA isolation went as expected and there is not much detail to give until the procedure is completed.
-Protein extraction results showed a high concentration of protein within the digestive gland. Something to compare
would be this concentration to the other tissue samples as I expect 530.27ug/ml to be a high concentration level.
Next Step:
-Complete RNA isolation procedures.
-Compare concentration levels from other groups in lab.
-Begin putting group project ideas together.
Reflection:
Purpose:
-To understand and perform basic fundamental techniques ( RNA isolation and Protein Extraction) used in molecular biology.
-The RNA isolation procedures in lab are used to measure the amount of mRNA being produced by the organism or the change in expression.
-The protein extraction procedure measures the concentration of protein being secreted by the organism.