November 27, 2013


Took gel and membrane off the rotary shaker that was in the fridge and followed the rest of the instructions for the Western blot that included a lot of washes and incubation time.

8. Rinse the membrane with 20 mL of water for 5 minutes, then decant. Repeat.
9. Incubate the membrane in 10 mL of Secondary Antibody Solution for 30 minutes. Decant.
10. Rinse the membrane in 10 mL of Antibody Wash for 5 minutes, then decant. Repeat 3 times.
11. Incubate the membrane in 10 mL of Secondary Antibody solution for 30 minutes. Decant.
12. Wash the membrane for 5 minutes with 20 mL of antibody wash, then decant. repeat 3 times.
13. Rinse the membrane with 20 mL of pure water for 2 minutes, then decant. Repeat twice.
14. Incubate the membrane in 5 mL of Chromogenic Substrate until a purple band appears. This will occur between 1-60 miinutes after adding the Chromogenic Substrate.
15. Dry the membrane on a clean piece of filter paper to the open air.

Our gel did not run right because nothing showed up on our gel and the membrane only had purple marks at the top, it did not extend down the membrane, not even the ladder was expressed.



November 26, 2013

Followed the SDS-PAGE protocol:

1. Boil water on hot plate
2. In a fresh, 1.5mL SCREW CAP tube add 15uL of your protein stock and 15uL of 2X Reducing Sample Buffer. Return your protein stock to the box in the -20C freezer labeled protein samples.
3. Mix sample by flicking tube. Centrifuge for 10 s to pool liquid in bottom of tube.
4. Boil sample for 5 minutes.
5. While the sample is boiling, observe assembly of gel box and gels. Rinse gel wells thourougly
6. When the sample is finished boiling, immediately centrifuge the tube for 1 min in order to pool the liquid.
7. Slowly load your sample into the appropriate well using a gel loading tip.
8. Put lid on gel box and plug electrodes into appropriate receptacles on the power supply.
9. Turn power supply on and set voltage to 150V. Run for 45 minutes.
10. Turn off power supply and disconnect gel box from power supply.
11. Remove lid from gel box.
12. Disengage the tension wedge.
13. Remove gel from gel box.
14. Carefully crack open cassette to expose gel.
15. Trim wells at top of gel.
16. Notch a designated corner of the gel to help you remember the correct orientation of the gel (which is the top/bottom of the gel, which is the right/left side of the gel)
17. Proceed to Western Blotting protocol

my samples were on the back plate, rows 1-6 on the gel.

We then proceded to the Western blot protocol:

1. Soak the filter paper, membrane and gel in Tris-Glycine Transfer Buffer for 15 minutes.
2. Assemble the blotting sandwich in the semi-dry blotting aparatus:
3. Transfer the blot for 30 minutes at 20V
4. Remove the gel from the sandwich and rinse off adhering pieces of gel with transfer buffer.
5. Wash the membrane 2 times, for 5 minutes each, with 20 mL of pure water.
6. Put the membrane in the plastic box and add 10 mL of Blocking Solution. Cover and incubate overnight on a rotary shaker set at 1 rev/sec.



November 25, 2013

Protein Quantification

quantified the protein samples to determine how much protein was present in each sample so that the same amount of protein is added to each well in the gel.

Sample
 Trial 1
 Trial 2
 Trial 3
 Avg.
A1-S
 1.218
 1.218
 1.218
 1.218
A2-S
 1.549
 1.549
 1.548
 1.55
A3-S
 1.556
 1.565
 1.555
 1.558
A4-S
 1.086
 1.084
 1.082
 1.084
A1-F
 1.391
 1.390
 1.388
 1.389
A2-F
 1.517
 1.516
 1.514
 1.515
A3-F
 0.911
 0.909
 0.909
 0.91
A4-F
 0.911
 0.91
 0.909
 0.91
Blank
 0
 0
 0
 0

back-calculated the protein concentration using the absorbance values and calculated the dilution for each so that the same amount of protein would go into each gel for the Western.
Sample
 Concentration
 Dilution
 ul to use
 dilute w/ water
A1-S
 17,404.3
 0.746
 11.19
 3.81
A2-S
 22,147
 0.586
 8.79
 6.21
A3-S
 22,375.7
 0.580
 8.70
 6.30
A4-S
 15,490
 0.838
 12.57
 2.43
A1-F
 19,847.1
 0.654
 9.81
 5.19
A2-F
 21,647.14
 0.60
 9
 6
A3-F
 12,990
 1
 15
 0
A4-F
 13,004.2
 0.999
 14.985
 0.05


Protein Quantification protocol

1. Labeled new tubes A1-4 S & F PBA GK 11/25/13
2. Dilute an aliquot of my protein samples 1:2 by pipetting 15uL of protein sample into each 2mL screw cap tube and then pipetting 15uL of DI water. Mix well by inverting.
3. In another 2 mL tube pipette 30uL of DI water (Blank)
4. to all tubes add 1.5mL of Bradford reagent.
5. Invert the tubes several times and then incubate at room temperature for 10 mins.
6. Transfer 1000uL blank to disposable cuvette.
7. Zero the spectrometer using the blank. Wipe the cuvette with a KimWipe first.
8. Transfer 1000uL of each sample to a disposable cuvette.
9. Measure the absorbance at 595nm three times for each sample and record the values.
10. Back calculate the protein concentration using the standard equation: Sample= (Avg. OD595 - 0.04+0.0403)/0.00007 (results shown above).



November 22, 2013

Protein Extraction:
1. Added 500 uL of CellLytic MT solution to each 1.5 mL snap cap tube containing my samples.
2. Homogenized the tissue with a sterile disposable pestle
3. Closed and inverted each tube several times.
4. Spun tubes for 10 mins at max speed in the refrigerated microfuge.
5. Labeled new tubes with A1-4: S (start) P (protein), and A1-4: F (finish) P (protein) and my initials GK and the date 11/22/13.

November 19, 2013

got primers in the mail, re-hydrated and Diluted primers (1 to 10 dilution). Labeled anemone primers : PE F, PE R. Labeled nudibranch primers: GK F, GK R.
Stored in top shelf of freezer in the lab.

November 13, 2013

We changed our experimental setup. Paul and I will be working with anemones and Jessica and Danny will be working with urchins. There will be 4 urchins and 4 anemones. 2 control and 2 treatment. Tissue samples will be taken before the first day of heat shock. 2 samples for each organism, one to measure gene expression using PCR and one to measure protein expression using a Western blot. Anemone tissue will be taken off the top of the organism, and urchin tissue will be taken by pulling off tube feet. Tissue samples will be put in centrifuge tubes will the following labels.

Control
 Treatment
Urchins
 Anemones
 Urchins
 Anemones
U1 G
 A1 G
 U3 G
 A3 G
U1 P
 A1 P
 U3 P
 A3 P
U2 G
 A2 G
 U4 G
 A3 G
U2 P
 A2 P
 U4 P
 A3 P

U= Urchin
A= Anemone
G= Gene expression
P= Protein expression.

Heat shock stress will be performed for 7 days, 2 hours a day with the experimental organisms under a heat lamp at 23oC. Tissue samples will also be taken on the last day of sampling.

November 12, 2013
Date
 11/12
 11/13
Day 1 Heat Shock
Sample collection
 11/14
Day 2
 11/15
Day 3
 11/16
Day 4
 11/17
Day 5
 11/18
Day 6
 11/19
Day 7
 11/20
Final heat shock, Final tissue sample
 11/22
Objective
 Determine plan of attack
 Set up experiment, how many organisms are being used, control and experimental, label tubes, collect tissue samples, put tissue samples on dry ice and then move to -80oC freezer, put experimental organisms under heat lamp for 2 hours.






start processing samples
Notes
Change in sample size and organisms used, only using anemones and urchins now. 4 urchins and 4 anemones, 2 control and 2 experimental for each.






Protein extraction.


November 5, 2013

Species collection Part 2

Went to Shilshole marina to collect green sea urchins for Jessica's project. Collected 17 green sea urchins off the side of the dock using nets and hands. Put the urchins in a bucket and took them back to the lab. The were put in a flow tank.


November 1, 2013

Species Collection

Went to Southern Alki beach at 930 am (low tide was at 950) in order to collect our organisms. Collected 5 shaggy mouse (Aeolidia papillosa) nudibranchs near a rocky jetty near some red algae. Also collected about 30 Anthropleura anemones and about 16 Pisaster sea stars. Put organisms in a bucket and took them back to the lab. Put each nudibranch in its own critter keeper (small plastic container with lid and little holes in each side) and labeled each container with N1-N5. Placed some red algae and Fucus in each container for food.

October 22, 2013

Summary

The purpose of this lab is to separate the extracted protein by its molecular weight using the process of SDS-PAGE. Then use the gel to perfom a Western blot to further examine gene expression.

Methods and Materials:

Protein Extraction and Analysis part 2

SDS - Polyacrylamide Gel Electorophoresis (SDS-PAGE)

Materials


Procedure

1. Boil water on hot plate
2. In a fresh, 1.5mL SCREW CAP tube add 15uL of your protein stock and 15uL of 2X Reducing Sample Buffer. Return your protein stock to the box in the -20C freezer labeled protein samples.
3. Mix sample by flicking tube. Centrifuge for 10 s to pool liquid in bottom of tube.
4. Boil sample for 5 minutes.
5. While the sample is boiling, observe assembly of gel box and gels. Rinse gel wells thourougly
6. When the sample is finished boiling, immediately centrifuge the tube for 1 min in order to pool the liquid.
7. Slowly load your sample into the appropriate well using a gel loading tip.
8. Put lid on gel box and plug electrodes into appropriate receptacles on the power supply.
9. Turn power supply on and set voltage to 150V. Run for 45 minutes.
10. Turn off power supply and disconnect gel box from power supply.
11. Remove lid from gel box.
12. Disengage the tension wedge.
13. Remove gel from gel box.
14. Carefully crack open cassette to expose gel.
15. Trim wells at top of gel.
16. Notch a designated corner of the gel to help you remember the correct orientation of the gel (which is the top/bottom of the gel, which is the right/left side of the gel)
17. Proceed to Western Blotting protocol

WesternBreeze Chromogenic Western Blot Immunodetection


Materials


Procedure

1. Soak the filter paper, membrane and gel in Tris-Glycine Transfer Buffer for 15 minutes.
2. Assemble the blotting sandwich in the semi-dry blotting aparatus:
3. Transfer the blot for 30 minutes at 20V
4. Remove the gel from the sandwich and rinse off adhering pieces of gel with transfer buffer.
5. Wash the membrane 2 times, for 5 minutes each, with 20 mL of pure water.
6. Put the membrane in the plastic box and add 10 mL of Blocking Solution. Cover and incubate overnight on a rotary shaker set at 1 rev/sec.
7. Decant liquid
8. Rinse the membrane with 20 mL of water for 5 minutes, then decant. Repeat.
9. Incubate the membrane in 10 mL of Secondary Antibody Solution for 30 minutes. Decant.
10. Rinse the membrane in 10 mL of Antibody Wash for 5 minutes, then decant. Repeat 3 times.
11. Incubate the membrane in 10 mL of Secondary Antibody solution for 30 minutes. Decant.
12. Wash the membrane for 5 minutes with 20 mL of antibody wash, then decant. repeat 3 times.
13. Rinse the membrane with 20 mL of pure water for 2 minutes, then decant. Repeat twice.
14. Incubate the membrane in 5 mL of Chromogenic Substrate until a purple band appears. This will occur between 1-60 miinutes after adding the Chromogenic Substrate.
15. Dry the membrane on a clean piece of filter paper to the open air.

October 15, 2013

Summary

The purpose of this lab is to prepare the reverse transcribed protein for qPCR to amplify a certain gene based on the primer added. The second purpose of this lab was to extract a protein for quantification. Techniques included: preparing a mastermix, adding a primer, adding the mastermix to wells of a white PCR plate, pippetting very small quantities.

Material and Methods:

Quantitative PCR

Materials



Procedure


1. Prepare mastermix for 5 reactions; 2 for control, 2 for the templates, and 1 to account for pippetting error.
2. Mastermix for 5 reactions contains; 62.5 uL SsoFast EvaGreen supermix (contains buffer, enzymes, dye, DNA polymerase, and nucleotides), 2.5 uL of upstream primer, 2.5 uL of downstream primer, and 52.5 uL of Ultra Pure Water. Primer that I used was for DNMT (DNA methyltransferase)
3. Add 24 uL mastermix to each well of a white PCR plate (4 wells total).
4. Thaw cDNA sample
5. Add 1 uL cDNA template to 2 of the wells that will be your template wells.
6. Add 1 uL of ultra pure water to the 2 negatve control wells.
7. cap the wells securely.
8. Label wells: GK 10/20/13
9. put wells on ice until they are loaded into the plate and the run begins

PCR conditions:
1. 95°C for 10 minutes
2. 95°C for 15s
3. 55 °C for 15 s
4. 72°C for 15 s (+ plate read)
5. Return to step 2 39 more times
6. 95°C for 10s
7. Melt curve from 65°C to 95°C, at 0.5°C for 5s (+plate read)

Protein Extraction

Materials



Procedure:

1. Obtain tissue sample; 34 g (gill tissue) Pac 35C (Pacific oyster)
2. label snap cap tube with your initials and date; GK Protein 10/15/13
3. Add 500 uL of CellLytic MT solution to the 1.5 mL snap cap tube containing your tissue
4. Homogenize the tissue with a sterile disposable pestle.
5. Close the tube and invert several times
6. Spin the tube in a refrigerated microfuge for 10 minutes at max speed.
7. While spinning, label a fresh tube with the word "Protein", source organism/ tissue, your initials, and today's date. GK Protein Pac C
8. Carefully transfer supernatant (the clear liquid on the top) to labeled tube and store tube on ice.

Conclusion: The next step would be to analyze the qPCR to measure the amplification of the gene DNMT. We could also do a Western blotting with our isolated protein in order to determine its molecular weight and shape.

Reflection:

The qPCR machine works by denaturing the cDNA with a certain temperature, then annealing the primer to a certain set of nucleotides with another temperature, then extending the product. This process is repeated 39 times in order to exponentially amplify the strand of cDNA that the primer attached to. Western blotting works by letting something (usually florescence) bind to the protein so you can see it.


October 8, 2013

Summary

The purpose of this lab was to develop techniques for extracting RNA from the tissue sample that we prepared last week. Also to get the RNA ready to do PCR by performing reverse transcription and creating cDNA. Techniques included pipetting, centrifuging, transferring only the aqueous layer to a new tube, homogenizing the sample, forming a pellet, incubating tube at a certain temperature.

Materials and Methods:

RNA extraction

Materials

Methods

1. Turn heating block on to 55oC
2. Incubate tube containing sample at room temperature for 5 minutes
3. add 200 uL of chloroform to sample under the fume hood and close tube securely
4. Vortex tube vigorously for 30s, if solution form a milky emulsion, you are vortexing correctly.
5. Incubate tube at room temperature for 5 minutes
6. Spin tube in refrigerated microfuge for 15 minutes at max speed.
7. Gently remove tube from microfuge.
8. Slowly and carefully transfer most of the aqueous phase (the top, clear portion) to a fresh tube labeled; RNA GK 10/8/13
9. Add 500 uL of isopropanol to the new tube and close the tube. Then invert the tube 8 times to mix the contents until the solution appears uniform.
10. Incubate at room temperature for 10 minutes.
11. Spin in refrigerated microfuge for 8 minutes at max speed.
12. After microfuge is complete, a small, white pellet (that contains the RNA) should be present at the bottom of the tube (usually dark in color).
13. Remove the supernatant from the tube and discard.
14. Add 1 mL of 75% EtOH to the tube containing your pellet. Close the tube and vortex briefly to dislodge the pellet from the side of the tube.
15. Spin in refrigerated microfuge at 7500g for 5 minuets.
16.Carefully remove supernatant. Make sure not the remove the pellet.
17. Briefly spin tube for about 15s to pool residual EtOH for final removal using a small pipette tip (P10 or P20 tips).
18. Leave the tube open and allow the pellet to dry at room temperature for no more than 5 minutes.
19. Re suspend pellet in 100uL of 0.1% DEPC-H2O by pipetting up and down and vortexing until pellet is dissolved.
20. Incubate tube at 55oC for 5 minutes to help solubilize the RNA.
21. Remove the tube for the heat and store on ice, this is your stock RNA that you can later quantify to tell the quality of your sample.

Reverse Transcription

Materials:



Methods:

1. Mix RNA sample by inverting the tube several times
2. In a 0.5 mL PCR tube labeled, GK cDNA, combine the following:
3. Incubate the mixture for 5 minutes at 70oC on the thermocycler then immediately transfer to ice. Briefly centrifuge the tube and add the following master mix:
4. Incubate the mixture for 1 hour at 42oC and then heat inactivate at 70oC for 30 min on the termocycler
5. Spin down the sample in a desk top centrifuge
6. Store on ice or at -20oC

Conclusion: The next step would be to take the cDNA and do PCR to examine gene expression.

Reflection:

RNA extraction is important for a number of different reasons. RNA can be used to look at gene expression, run PCR, look at polymerism, and to amplify certain strands for further examination. Reverse transcription is used to convert the RNA into cDNA which is more stable and can be amplified using PCR to look at gene expression.



October 1 2013

Summary

The purpose of this lab was to develop techniques for extracting DNA from tissue samples. The tissue samples used were from Olympic or Pacific oysters and were either mantle or gill tissue. I worked with mantle tissue from Olympic oysters for my DNA extraction and mantle tissue from a Pacific oyster for my RNA extraction. Techniques used included pipetting, centrifuging, homogenizing samples, using Tri-Reagent, washing sample in ethanol, and quantifying the DNA that was extracted.

Materials and Methods:

RNA extraction Part 1

Materials

Methods

1. Label snap cap tube containing tissue sample with initials and the date using a lab marker. 32, M Pac, GK, 10/1/13. (M= Mantle, Pac=Pacific)
2. Add 500uL of TriReagent to the 1.5mL snap cap tube containing tissue under the fume hood. Store on ice.
3. Carefully homogenize the tissue using a disposable pestle.
4. After the sample is completely homogenized, add an additional 500uL of TriReagent to the tube under the fume hood and close the tube tightly.
5. Vortex vigorously for 15s.
6. Store sample at -80C.


DNA Isolation


Materials


Methods

1. Label snap cap tube with DNA sample, initials, and date and store on ice. 80m, Oly, GK, 10/1/13.
2. Add .5mL DNazol to DNA sample tube. Homogenize tissue using sterile pestile. After tissue is homogenized, add .5mL more of DNazol and mix well.
3. Let sample incubate at room temperature for 5 minutes.
4. Spin sample at 10,000 x g (room temp) for 10 minutes.
5. Transfer supernatant to a new, labeled tube. GK, DNA, 10/1/13.
6. Add .5mL of 100% ethanol to sample.
7. Mix sample by inverting tube 5-8 times.
8. Store sample at room temperature for 1 minute.
9. Your DNA should form a cloudy precipitate. If no precipitate forms centrifuge tube at 5,000 x g for 5 min so that DNA will form a pellet. Remove lysate (liquid that is not DNA) and discard, keeping the DNA pellet in the tube.
10. Let sample sit at room temperature for 1 minute.
11. Wash DNA with 1 mL of 75% ethanol: Pipette the ethanol into your DNA tube, invert 6 times, and let sit for 1 minute. Remove ethanol from tube and repeat.
12. If there is ethanol left at the bottom of your tube after the second wash, remove with a small pipette.
13. Add 300 uL of 0.1% DEPC water to your DNA and pipette up and down multiple times to dissolve. Also use vortex to further dissolve.
14. Bring DNA sample to Nanodrop to quantify.

DNA Quantification

Methods

  1. Pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal and lower the arm.
  2. Select "dsDNA" from the pulldown menu
  3. Click "Blank", to zero the instrument. NOTE: steps 1 and 2 only need to be done once for the whole class.
  4. Pipette 2µL of your DNA sample onto the Nanodrop pedestal and lower the arm
  5. Click "Measure". Record your DNA concentration (ng/µL), A260/280 ratio and A260/230 ratio. NOTE: The Nanodrop uses the Beer-Lambert Law to calculate DNA concentration for you.
  6. Raise the arm and wipe off you sample with a KimWipe
  7. Clearly label your stock DNA sample with the word "DNA", source organism/tissue, your initials, today's date and the concentration in ug/uL.
  8. Store sample at -20ºC.

Results

A260/280 ratio= 1.91
A260/230 ratio= 0.66

Conclusion

The data indicated that my DNA was good quality because my A260/280 ratio is 1.91 and purified DNA has a A260/A280 ratio of 1.7-1.9. This was as expected and I was able to extract a lot of DNA out of my sample. The next step would be to run the DNA in a gel electrophoresis or use it as a primer to isolate a specific fragment for PCR.

Reflection

The purpose of this lab was to develop important lab techniques that form the basis for doing more in-depth molecular tests. Learning how to extract DNA and RNA is essential if you want to find out how the environment affects an organism's gene expression. The procedures performed in lab are used to extract DNA and then measure the quality of your DNA that was extracted so that you can use it to run further tests. These methods could be used to study gene expression by using PCR. I was unclear about how the Nanodrop worked and what the ratio meant that we measured.

Genes:

AFGP- Antifreeze glycoprotein- used to permit the survival of arctic fish in subzero environments.

Norepinephrine- stress hormone