We extracted protein and rean a argros gell and western blot.
Experiment ran for 2 weeks after each week once on novebmer 12th and once on november 19th we took tissue samples of the gills of one mussel from each treatment group.



Experimental set up

20131112_143502.jpg LAB 6

we took initial samples of tissue from two different mussles to use as a baseline in the rest of our experiment.

Lab 5


Today in lab as a group we discussed what we would be doing for our projects.
As a group we got approval for our project.
LAB 4

Objectives


ELECTROPHORESIS PROCEDURE
  1. Place gel in gel box and fill with 1x TAE buffer (to fully cover wells)
  2. Remove combs from wells
  3. Load 7uL 100bp ladder in far left lane
  4. Load 20uL of your PCR sample into the gel (retain the remaining vol at -20ºC)
  5. Run gel at ~ 100V for ~ 40min
  6. Visualize the gel on the UV transilluminator
  7. Mine was in well 4

Protein Extraction and Analysis Part 2


  1. Have water boiling
  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. Briefly centrifuge (10s) to pool liquid in bottom of tube.
  4. Boil sample for 5 mins.
  5. While sample is boiling, observe assembly of gel box and gels. Rinse gel wells thoroughly as demonstrated.
  6. When sample is finished boiling, immediately centrifuge for 1min. to pool liquid.
  7. Slowly load your entire 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 45mins. CHECK YOUR AGAROSE GEL RESULTS. MAKE SURE EVERYTHING IS SET UP FOR WESTERN BLOT.
  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 (i.e. which is the top/bottom of the gel, which is the right/left side(s) of the gel)
  17. Proceed to Western Blotting protocol.


WesternBreeze Chromogenic Western Blot Immunodetection


Western Blot Protocol
This is a lengthy protocol with many incubation steps. There will be one gel for the entire class. As a class, you should assign yourselves steps so that everyone can participate and so that we don't waste time. When it is not your turn to attend to the gel, you can do protein and RNA extractions for your project.
  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 appartus:
    1. Anode (+++)
    2. filter paper
    3. membrane
    4. gel
    5. filter paper
    6. cathode (---)


  1. Transfer the blot for 30 minutes at 20V
  2. Remove the gel from the sandwich and rinse off adhering pieces of gel with transfer buffer.
  3. Wash membrane 2 times, for 5 minutes each, with 20 mL of pure water.
  4. 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 revolution/second.
  5. Your TA will do the rest of the steps. After class tomorrow you can come and see your results.
  6. Decant liquid.
  7. Rinse the membrane with 20 mL of water for 5 minutes, then decant. Repeat.
  8. Incubate the membrane in 10 mL of Primary Antibody Solution. Decant the solution.
  9. Rinse the membrane with 20 mL of Antibody Wash for 5 minutes, then decant. Repeat 3 times.
  10. Incubate the membrane in 10 mL of Secondary Antibody Solution for 30 minutes. Decant.
  11. Wash the membrane for 5 minutes with 20 mL of Antibody wash, then decant. Repeat 3 times.
  12. Rinse the membrane with 20 mL of pure water for 2 minutes, then decant. Repeat twice.
  13. Incubate the membrane in 5 mL of Chromogenic Substrate until a purple band appears. This will occur between 1-60 minutes after adding the Chromogenic Substrate.
  14. Dry the membrane on a clean piece of filter paper to the open air.




LAB 3


Supplies and Equipment:


Procedure background

A reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribed single-stranded RNA into double-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of this. The resulting cDNA is more stable than RNA (which degrades quickly) can be used for downstream applications such as measuring gene expression. Partial nucleotide sequences of cDNAs are often obtained as expressed sequence tags. (This text has been adapted from Wikipedia)

REVERSE TRANSCRIPTION PROTOCOL

  1. Mix your stock RNA sample by inverting tube several times.
  2. In a 0.5 ml PCR tube labeled with your initials and “cDNA” combine the following:
    1. 5 μl of YOUR total RNA (extracted and quantified in lab)
    2. 1 μl of oligo dT
    3. 4 μl of nuclease free H2O

  1. Incubate the mixture for 5 min at 70°C on the thermocycler then immediately transfer to ice. Briefly centrifuge you tube and the add the following:
    1. 5 μl of M-MLV 5X Reaction Buffer
    2. 5 ul of dNTPs
    3. 1 μl of M-MLV RT
    4. 4 μl of nuclease free H2O

  1. Incubate the mixture for 60 min at 42°C and then heat inactivate at 70°C for 3 min on the thermocycler.
  2. Spin down the sample in a desk top centrifuge.
  3. Store on ice or at -20°C



Quantitative PCR



Supplies and Equipment:


qPCR PROCEDURE

You will run each template (cDNA) in duplicate in addition to two negative controls (no template) - calculate how many reactions this will be!

1. Prepare master mix: Prepare enough master mix for your number of reactions +1 to ensure sufficient volume recovery.

Master Mix: 62.5 uL
upstream primer 2.5uL
downstream primer2.5uL
ultra pure water52.5uL


2. Add mastermix to wells of a white PCR plate
3. Thaw cDNA samples.
4. Add 1uL cDNA template to each reaction.
5. Add 1uL of ultra pure water to the negative control wells.
6. Cap the wells securely.
7. If necessary, spin the strips to collect volume in the bottom of the wells.
8. Ensure the lids are clean and place strips on ice. (I like to wipe the lids with a clean kimwipe)
9. Load the plate, verify the PCR conditions and start the run (this will be done by your TA).

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 and Analysis Part 1



Supplies and Reagents




Procedure Background

This section provides an explanation of the methods being used and provieds some essential background information.




PROTEIN EXTRACTION PROTOCOL

  1. Record the weight of your tissue that has been denoted on the tube.
  2. Label the snap cap tube containing your tissue sample with your initials and the date using a lab marker.
  3. Add 500 ul of CellLytic MT solution to the 1.5mL snap cap tube containing your cut piece of frozen tissue.
  4. Homogenize the tissue with a sterile disposable pestle.
  5. Close the tube and invert the tube several times.
  6. Please find a few other people at or near this same stage and form a group for this step. Spin the tube in a refrigerated microfuge for 10 mins at max speed.
  7. While spinning, label a fresh tube with the word "Protein", source organism/tissue, your initials, and today's date.
  8. Carefully transfer supernatant (the clearish liquid on top) to labeled tube and store tube on ice.



PROTEIN QUANTIFICATION PROTOCOL

  1. Lable a fresh 2 mL screw cap tube withe the word "Protein", BA (for Bradford Assay), your initials, and today's date.
  2. Dilute an aliquot of your protein sample 1:2 by pipetting 15uL of your protein sample into the 2 mL screw cap tube and the pipetting 15uL of DI water. Mix well by pipetting. Note: this dilution step is performed to ensure the sample absorbance falls within the range of the standard curve
  3. In a second 2 mL tube pipette 30uL of DI water (this tube will serve as your blank). Label tube as 'blank'
  4. To both tubes add 1.5mL of Bradford reagent. Tip: Pippet 1000ul of reagent into each tube and then pipet another 500 ul of reagent into each tube for a total of 1500u ul or 1.5 mL.
  5. Invert the tubes several times and then incubate at room temperatire (RT) for 10mins.
  6. Mix the 'blank' via pippeting and transfer to a 1000ul to a plastic, disposable cuvette.
  7. Zero the spectrophotometer using your blank sample. Be sure to wipe the cuvette with a KimWipe first as any fingerprints or smudges can alter the reading.
  8. Mix the 'sample' via pippeting and transfer 1000 ul to a plastic, disposable cuvette
  9. Measure the absorbance at 595nm and record the value. Be sure to wipe the cuvette with a KimWipe first as any fingerprints or smudges can alter the reading.
  10. Remove the cuvette from the spectrophotometer. Using a P1000 set to 1000 ul, carefully pipette the solution in the cuvette up and down a couple of times to mix.
  11. Measure the absorbance at 595nm and record the value a second time.
  12. Average the two absorbance values you recorded.
  13. Back-calculate your protein concentration using the standard curve below. Hint: Use the equation on the graph provided. The relationship between absorbance and concentration is linear and defined by the equation y=mx+b. You have the average absorbance of your sample, x, and you want to calculate the concentration, y. Don't forget to account for the dilution in step 2!
  14. Give your protein sample to the TA for storage at -20ºC.
x


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LAB 2


For a fun introduction to quantification using the Nanodrop please watch this video.

RNA EXTRACTION PROTOCOL
Continued from Lab 1

1. Turn on heating block to 55°C.
2. Incubate your homogenized tissue sample (from Lab 1) tube at room temperature (RT) for 5 mins.
3. In the fume hood, add 200uL of chloroform to your sample and close the tube. NOTE: Due to the high volatility of chloroform, pipetting needs to be done carefully and quickly. Have your tube open and close to the container of chloroform before drawing and chloroform into your pipette tip.
4. Vortex vigorously for 30s. You are vortexing correctly if the solution becomes a milky emulsion.
5. Incubate tube at RT for 5 mins.
6. Spin tube in refrigerated microfuge for 15 mins. @ max speed.
7. Gently remove tube from microfuge. Be sure not to disturb the tube.
8. Slowly and carefully transfer most of the aqueous phase (the top, clear portion) to a fresh microfuge tube. Do NOT transfer ANY of the interphase (the white, cell debris between the aqueous and organic phase).
9. Close the tube containing the organic and interphase and properly dispose of the liquid inside the tube as well as the tube itself at the end of the lab.
10. Add 500uL isopropanol to the new tube containing your RNA and close the tube.
11. Mix by inverting the tube numerous times until the solution appears uniform. Pay particular attention to the appearance of the solution along the edge of the tube. If mixed properly, it should no longer appear viscous/"lumpy".
12. Incubate at RT for 10 mins.
13. Spin in refrigerated microfuge at max speed for 8 mins. When placing your tube in the microfuge position the tube hinge pointing up, away from the center of the microfuge.
14. A small, white pellet (RNA and salts) should be present. If not, do not fret an continue with the procedure.
15. Remove supernatant.
16. Add 1mL of 75% EtOH to pellet. Close tube and vortex briefly to dislodge pellet from the side of the tube. If the pellet does not become dislodged, that is OK.
17. Spin in refrigerated microfuge at 7500g for 5mins.
18. Carefully remove supernatant. Pellet may be very loose. Make sure not to remove pellet!
19. Briefly spin tube (~15s) to pool residual EtOH.
20. Using a small pipette tip (P10 or P20 tips), remove remaining EtOH.
21. Leave tube open and allow pellet to dry at RT for no more than 5mins.
22. Resuspend pellet in 100uL of 0.1%DEPC-H2O by pipetting up and down until pellet is dissolved.
23. Incubated tube at 55C for 5mins. to help solubilize RNA.
24. Remove tube from heat, flick a few times to mix and place sample on ice. This will be your stock RNA sample.
25. Quantitate RNA yield using Nanodrop spectrophotometer.

RNA QUANTIFICATION
NOTE: Always keep your RNA samples on ice!
1. Pipette 2µL of 0.1%DEPC-H20 onto the Nanodrop pedestal and lower the arm.
2. Click "Blank", to zero the instrument. NOTE: steps 1 and 2 only need to be done once for the whole class.
3. Pipette 2µL of your RNA sample onto the Nanodrop pedestal and lower the arm
4. Click "Measure". Record your RNA concentration (ng/µL), A260/280 ratio and A260/230 ratio. NOTE: The Nanodrop uses the Beer-Lambert Law to calculate RNA concentration for you. See Lab 1 notes on RNA extraction for more information on the calculation and how to evaluate RNA purity using A260/280 and A260/A230 ratios.
6. Raise the arm and wipe off you sample with a KimWipe
7. Clearly label your stock RNA sample with the word "RNA", source organism/tissue, your initials, today's date and the concentration in ug/uL.
8. Give your samples to the TA for storage at -80C.

EVERSE TRANSCRIPTION PROTOCOL

  1. Mix your stock RNA sample by inverting tube several times.
  2. In a 0.5 ml PCR tube labeled with your initials and “cDNA” combine the following:
    1. 5 μl of YOUR total RNA (extracted and quantified in lab)
    2. 1 μl of oligo dT
    3. 4 μl of nuclease free H2O

  1. Incubate the mixture for 5 min at 70°C on the thermocycler then immediately transfer to ice. Briefly centrifuge you tube and the add the following:
    1. 5 μl of M-MLV 5X Reaction Buffer
    2. 5 ul of dNTPs
    3. 1 μl of M-MLV RT
    4. 4 μl of nuclease free H2O

  1. Incubate the mixture for 60 min at 42°C and then heat inactivate at 70°C for 3 min on the thermocycler.
  2. Spin down the sample in a desk top centrifuge.
  3. Store on ice or at -20°C







Lab 1

RNA Extractions part 1

Supplies and reagents
  1. Micropipettes (1- 1000uL)
  2. sterile filter pipette tips (1- 1000uL)
  3. sterile (RNase free) 1.5 mL microcentrifuge tubes
  4. sterile disposeable pestiles
  5. vortex
  6. ice buckets
  7. cloves
  8. lab pens
  9. safety glasses
  10. triReagent
Procedure
Steps
DNA Isolation (DNazol)- procedure
Results
the results where
Sample 6
ABS 8.757
lambda 230
A-260 4.240
A-280 2.198
260/ 280 1.93
260/230 .48
212.0 ng/ul