Summary
Preparation of agarose gel, electrophoresis of PCR products
Procedure
Agarose Gel Preparation
1. Mixed 2g agarose with 150mL 1x TAE in a 500mL flask.
2. Microwaved solution for 2:15.
3. Swirled to mix and prevent boiling over.
4. Microwaved additional 45 seconds.
5. Cooled solution for ~5 minutes.
6. Mixed 12 µL ethidium bromide into solution.
7. Poured solution into gel tray.
8. Added gel combs and allowed gel to cool and set for ~60min.
PCR Product Electrophoresis
1. Placed pre-stained agarose gel in gel electrophoresis box and removed combs.
2. Covered gel with 1x TAE buffer.
3. Loaded 20µL 100bp ladder in well #1 of the bottom row of wells.
4. Loaded PCR products from 4 Dec 2009 in wells.
5. Ran gel at 100V for 50min hour.
6. Observed bands using UV transilluminator.
Results
Gel Image
- Band at ~400bp in Na+/K+-ATPase from Sample E.
- Primer-dimer bands in all Na+/K+ATPase wells.
- Bands at ~630bp and ~590bp in TreE3 wells from Samples A, B and E.
- Primer-dimer bands in all TreE3 wells
- Primer-dimer bands in all TreE2 wells
Conclusions
The band at ~630bp for the TreE3 primer PCR reaction is indicative of expression of Trehalase-3 at all times, which was expected. The slightly smaller band at ~590bp could be indicative of Trehalase-2, since there is a 99.7% overlap in the gene sequence between Trehalase-3 and Trehalase-2. Therefore, it is possible that the Trehalase-3 cDNA reaction also resulted in amplification of Trehalase-2 cDNA. The clear band at ~400bp for Na+/K+-ATPase is consistent with the expected 407bp amplicon, indicating expression of Na+/K+-ATPase at sampling time E.
The fluorescence in all qPCR plate wells from 2 December is likely a result of primer-dimer formation, since it occurred in all of the reactions.
Next Steps
If I were to continue with this project, I would redesign the primers to not be reverse complimentary and re-run the qPCR.
4 December 2009
Summary
Preparation of PCR reactions, running PCR reactions
Procedure
Preparation of PCR Reactions
1. Prepared PCR master mix for each primer (Tre2, Tre3, Na+/K+-ATPase) in sufficient volume for five 25µL reactions:
- 62.5µL GoTaq Green Master Mix, 2X
- 6 µL 10µM upstream primer
- 6 µL 10µM downstream primer
- 50.5 µL DNAse/RNAse free H2O
2. Added 24µL of PCR master mix to PCR tubes:
X* Tubes - Tre2
Y* Tubes - Tre3
Z* Tubes - Na/K
3. Added template 1µL cDNA (from 12/1/09) or DNAse free H2O to tubes:
*A Tubes - cDNA (from A5)
*B Tubes - cDNA (from B5)
*E Tubes - cDNA (From E5)
*- Tubes - DNAse free H2O
4. Placed tubes in thermalcycler for forty (40) amplification cycles:
a. 95ºC - 30 sec (Denaturation)
b. 55ºC - 30 sec (Annealing)
c. 72ºC - 90 sec (Extension)
20. Final extension at 72ºC for 3 minutes.
21. PCR products stored at 4ºC.
Results
Summary of Reactions Prepared
XA: Tre2/A5
XB: Tre2/B5
XE: Tre2/E5
X-: Tre 2/H2O
YA: Tre3/A5
YB: Tre3/B5
YE: Tre3/E5
Y-: Tre3/H2O
ZE: NaK/E5
Z-: NaK/H2O
There was not enough template cDNA remaining to run NaK/A5 or NaK/B5.
Next Steps
Agarose gel preparation and electrophoresis of PCR reaction products will be conducted on 7 December 2009.
2 December 2009
Summary
qPCR Results from 1 December 2009
Results and Conclusions
The qPCR fluorescence and melting curves were confounding. Early amplification was present in many of the negative controls for all primers, and the peak log fluorescence for TreE2 and TreE3 reactions was 0.5, well below a 'normal' observation if amplification occurred. Na+/K+-ATPase reactions had consistent peaks, which appeared earlier than one of the negatives (cycle 25 versus cycle 31), but well after the other negative (cycle 19).
All of the curves were very noisy and interpretation of results was a shot-in-the dark at best. It is likely that either:
1. There was contamination in the water used for all reactions.
2. Primer dimer formation occurred for all samples, possibly leading to more rapid amplification in the blanks where primers wouldn't be annealing to cDNA sequences.
3. The annealing temperature (50ºC) may have been too low due to the large amplicon sizes for all three products (407-630bp).
Next Steps
Traditional PCR with a higher annealing temperature to check for any expression of genes of interest, primer dimer formation or possible contamination.
-
1 December 2009
Summary
Standardization of RNA concentration, reverse transcription of RNA to form template cDNA, qPCR.
Procedure
RNA Standardization
1. Thawed DNAsed RNA samples (from 11/23).
2. Added 1.9µL 0.1% DEPC-H2O to B4 to standardize to A4.
Reverse Transcription
1. Mixed RNA sample by inverting tube.
2. Added 5µL sample to clean PCR tubes (A5, B5, C5, D5, E5).
3. Incubated tubes in thermal cycler 75ºC for 5 minutes
4. Transferred tubes to ice and allowed sample to cool for 5 minutes.
5. Added to sample tubes:
-4µL 5x AMV RT Buffer
-8µL dNTPs
-1µL Oligo dT Primer
-1µL AMV RTranscriptase
-1µL RNase free H2O
6. Vortexed and spot-spinned to mix.
7. Incubated at room temperature for 10 minutes.
8. Incubated for 1hr at 37ºC in thermocycler to transcribe cDNA.
9. Heat inactivated in thermal cycler for 3 minutes at 95ºC.
10. Spot-spinned sample tubes.
11. Stored on ice.
qPCR
1. Prepared qPCR master mixes for 23x (22 + 1 for additional volume) 50µL reaction in a 1.5mL snap cap tube for each primer (Trehalase 2, Trehalase 3, Na/K-ATPase):
- 575µL Immomix Master Mix, 2X
- 92µL 50µM Syto-13 dye
- 115µL 10µM upstream primer (Prepared 11/17/09)
- 115µL 10µM downstream primer (Prepared 11/17/09)
- 207µL DNAse/RNAse free H2O
2. Prepared qPCR reactions on qPCR plates:
- Two negative controls for each primer = 3 x 2 = 6
- Two RNA controls for each sample (A-E), for each primer = 5 x 2 x 3 = 30 total
- Two cDNA reactions for each sample (A-E), for each primer = 5 x 2 x 3 = 30 total
3. Centrifuged qPCR plates briefly to pool reactants.
4. Wiped lids with a KimWipe.
5. Loaded qPCR plates in Opticon machine (reaction link ).
6. qPCR run in Opticon machine using settings:
a) Incubate at 95ºC for 10 minutes
b) Incubate at 95ºC for 30 seconds
c) Plate Read
d) Incubate at 72ºC for 30 seconds
e) Plate Read
f) Repeat b-e 39 times
g) Incubate at 95ºC for 1 minute
h) Incubate at 55ºC for 1 minute
-Manual Ramp Rate: 0.2ºC per second
i) Melting curve from 55ºC to 95ºC, read every 0.5ºC
j) Incubate at 21ºC for 10 minutes
24 November 2009
Summary
Continuation of RNA isolation from samples, DNAsed RNA samples, RNA quantification.
Procedure
RNA Isolation (Part 2 - continuation from 11/16-11/23)
1. Thawed RNA samples (A2, B2, C, D2, E2) at room temperature.
2. Added 200µL chloroform to sample tubes.
3. Vortexed samples for ~30s.
4. Incubated at room temperature for ~5 minutes.
5. Microfuged in refrigerator at maximum speed for ~15 minutes.
6. Transferred aqueous phase to fresh RNAase free 1.5µL sample tube.
7. Added 500µL isopropanol to sample tube.
8. Mixed by inversion and incubated at room temperature for ~10 minutes.
9. Microfuged in refrigerator at maximum speed for 8 minutes.
10. Supernatant removed from sample.
11. Added 1mL 75% EtOH to sample tube, vortexed to loosen pellet.
12. Microfuged in refrigerator at 7500g for 5 minutes.
13. Removed supernatant.
14. Microfuged for an additional 15 seconds.
15. Removed remaining supernatant, and allowed pellet to dry for ~3 minutes.
16. Added 100µL 0.1% DEPC-H2O to sample tube.
17. Dissolved pellet by mixing with pipette.
18. Incubated in 55ºC water bath for ~5min.
19. Flicked tube to mix.
20. Stored on ice.
DNAse Treatment of Samples
1. Added 2.5µL of Turbo DNAse Buffer to 0.5mL snap cap tubes (A3, B3, C3, D3, E3).
2. Added 1.0 µL of Turbo DNAse to each tube.
3. Added 20.5µL of sample RNA (from A2, B2, C, D2, E2) to tubes.
4. Incubated tubes for 30 min at 37ºC.
5. Added 1.0µL of Turbo DNAse to each tube.
6. Incubated tubes for 30 min at 37ºC.
7. Added 2.5µL DNAse inactivation reagent to each tube.
8. Mixed by flicking and inversion.
9. Incubated at room temperature for 2 minutes.
10. Refrigerated microfuged tube at 10000g for 90 sec.
11. Transferred 18µL supernatant to clean 0.5mL tubes (A4, B4, C4, D4, E4).
RNA Quantification with Nanodrop (Repeated for A4, B4, C4, D4, E4)
1. Zeroed Nanodrop with 2µL 0.1% DEPC-H2O (only once for all samples)
2. Pipetted 2µL sample on Nandrop pedestal.
3. Measured absorbance and concentration at 230nm, 260nm and 280nm (printed report)
4. Stored RNA sample at -80ºC.
Results
RNA Quantification
| Sample |
RNA Concentration (µg/µl) |
A260 |
A280 |
260/280 Ratio |
260/230 Ratio |
| A4 |
120.15 |
3.004 |
1.571 |
1.91 |
1.09 |
| B4 |
132.31 |
3.308 |
1.680 |
1.97 |
1.48 |
| C4 |
3.02 |
0.076 |
0.085 |
0.89 |
0.13 |
| D4 |
8.76 |
0.219 |
0.153 |
1.44 |
0.26 |
| E4 |
9.22 |
0.231 |
0.165 |
1.40 |
0.41 |
Conclusions
The nanodrop results indicate that all RNA samples are clean, however, the concentrations for C4, D4 and E4 are very low and could lead to qPCR difficulties down the line. The concentrations of D4 and E4 are too low to reasonably dilute, although the concentration of B4 should be standardized to A4 before continuing to reverse transcription. The low RNA concentration for C4 isn't surprising since it is from the batch of cysts that did not hatch.
Next Steps
1. Standardization of B4 to the concentration of A4.
2. Reverse transcription to create template cDNA samples.
3. qPCR of sample cDNA.
16-23 November 2009
Summary
Restarted hatchery, sampled Artemia franciscana throughout reanimation, RNA isolation.
Procedure
Artemia Hatchery Prepartion
(Procedure same as 11/10-11/16/09, with a new sample stock)
Sampling Procedure for Cysts (Samples A2 & B2)
1. Extracted concentrated cysts from bottom of tank in a 1.5mL snap cap tube.
2. Centrifuged briefly at maximum speed to pool cysts at bottom of tube.
3. Removed excess seawater using P200 micropipette.
4. Added 500µl TriReagent to sample tube and homogenized cyst tissue with pestle.
5. Added 500µl TriReagent to sample tube and vortexed for 15s.
6. Stored sample at -80ºC.
Sampling Procedure and RNA Isolation Part 1 for Nauplii (Samples D2 & E2)
1. Turned off air supply.
2. Placed dissecting microscope light source against exterior wall of the hatchery.
3. Allowed 3 minutes for phototaxic response by nauplii.
4. Extracted ~5ml of high nauplii density seawater from near light source and added ~1ml to each of five 1.5ml snap cap tubes.
5. Terminated nauplii using pestle.
6. Microfuged at maximum speed for 2 minutes to pool tissue.
7. Extracted tissue pellet from each tube and transferred to clean 1.5ml snap cap sample tube.
8. Microfuged sample tube briefly at maximum speed.
9. Removed excess seawater with P20 micropipette.
10. Added 500µl TriReagent to sample tube and homogenized nauplii tissue with pestle.
11. Added 500µl TriReagent to sample tube and vortexed for 15s.
12. Stored sample at -80ºC.
Sampling Timeline
11/16/09 - 16:25 - New cysts added to hatchery
11/16/09 - 16:45 - Sample Tube A2 (cysts)
11/17/09 - 09:50 - Sample Tube B2 (cysts)
11/18/09 - 10:30 - Sample Tube D2 (nauplii)
11/23/09 - 14:45 - Sample Tube E2 (nauplii)
Primer Preparation
1. Centrifuged dehydrated primers
2. Added 10µL of DNAse/RNAse free H2O per nM dehydrated primer.
3. Transferred 150µL of 10µM primer solution to clean 1.5µL RNAse/DNAse free snap cap tubes.
4. Stored primers at -20ºC.
Results
The cysts hatched <32 hours after being added to the tank.
Prepared Primers:
- 150µL 10µM Artemia franciscana Trehalase 2 Forward
- 150µL 10µM Artemia franciscana Trehalase 2 Reverse
- 150µL 10µM Artemia franciscana Trehalase 3 Forward
- 150µL 10µM Artemia franciscana Trehalase 3 Reverse
- 150µL 10µM Artemia franciscana Na/K ATPase Forward
- 150µL 10µM Artemia franciscana Na/K ATPase Reverse
Conclusion
The cysts hatching indicates that the previous batch was ineffective.
Next Steps
Continue with RNA extraction protocol, reverse transcription and qPCR. Tube C from the first hatchery attempt will be included in future procedures to check for any gene expression indicating viable cysts.
10-15 November 2009
Summary
Began research project on Artemia spp. trehalase and Na/K-ATPase gene expression during reanimation of cysts, preliminary samples.
Procedure
Artemia Hatchery Preparation
1. Rinsed and dried prefabricated Artemia hatchery using tap water.
2. Added seawater to fill to within 7" hatchery brim.
3. Attached airstone to bottom of tank for continuous oxygenation.
4. Added an unmeasured volume of of Artemia cysts to hatchery.
5. Directed upward-facing dissection scope lamps on hatchery.
Sampling Procedure and RNA Isolation Step 1
1. Extracted ~4ml of hatchery water (with suspended cysts) and added ~1ml each to 1.5ml snap cap tubes.
2. Centrifuged snap cap tubes at maximum speed for 2 minutes.
3. Determined mass of a clean 1.5ml snap cap tube (Sample Tube [Letter]).
4. Using P1000 micropipettor, transferred cysts at bottom of each centrifuged tubes to Sample Tube [Letter].
5. Centrifuged Sample Tube [Letter] briefly to pool contents at bottom of tube.
6. Used a small bore P200 micropipettor to remove excess supernatant (seawater) from Sample Tube [Letter].
7. Determined mass of Sample Tube [Letter] to find wet weight of sample.
8. Added 500µl TriReagent to Sample Tube [Letter] and homogenized with pestle.
9. Vortexed Sample Tube [Letter] for 15 seconds.
10. Stored Sample Tube [Letter] at -80ºC.
Sampling Occasions
Artemia Hatchery Preparation Conducted:
11/10/09 - 14:00
Sampling Procedure and RNA Isolation Step 1 Was Conducted:
11/10/09 - 14:35 (Sample Tube A)
11/11/09 - 10:05 (Sample Tube B)
11/13/09 - 8:40 (Sample Tube C)
Results
Sample A (Cysts) Mass: 90mg wet weight
Sample B (Cysts) Mass: 52mg wet weight
Sample C (Cysts) Mass: 19mg wet weight
The cysts were expected to hatch within 18-72 hours, but did not hatch after nearly 146 hours.
Conclusions
Since the cysts didn't hatch, it is likely that they had exceeded their shelf-life. Another hatchery prepared using the same batch of cysts also didn't hatch.
Next Steps
Restart hatchery procedure using a new batch of cysts, obtain fresh samples.
3 November 2009
Summary
Quantitative PCR was performed to quantify expression of tropomyosin in Argopecten spp. adductor muscle tissue.
Procedure
1. Prepared qPCR master mix for 50µL reaction in a 1.5mL snap cap tube:
- 175µL Immomix Master Mix, 2X
- 28µL 50µM Syto-13 dye
- 17.5µL 10µM upstream primer (Prepared 10/20/09)
- 17.5 µL 10µM downstream primer (Prepared 10/20/09)
- 98µL DNAse/RNAse free H2O
2. Prepared 24µL 1:3 dilution RNA from template RNA (Prepared 10/13/09) and DNAse/RNAse free H2O, in a PCR tube.
3. Added 48µL qPCR master mix to qPCR plate, wells A, B, C, D, E and F.
4. Thawed and added 2µL cDNA sample (Prepared 10/20/09) to qPCR wells A and B.
5. Added 2µL DNAse/RNAse free H2O to qPCR wells C and D (negative control)
6. Added 2µL 1:3 dilution RNA to qPCR wells E and F to test for genomic carryover.
7. Capped qPCR wells and spun briefly to pool reactants in bottom of wells.
8. Wiped lids with a Kim Wipe.
9. Loaded in column 6, rows A-F of PCR plate.
10. qPCR run in Opticon machine using settings:
a) Incubate at 95ºC for 10 minutes
b) Incubate at 95ºC for 30 seconds
c) Plate Read
d) Incubate at 72ºC for 30 seconds
e) Plate Read
f) Repeat b-e 39 times
g) Incubate at 95ºC for 1 minute
h) Incubate at 55ºC for 1 minute
-Manual Ramp Rate: 0.2ºC per second
i) Melting curve from 55ºC to 95ºC, read every 0.5ºC
j) Incubate at 21ºC for 10 minutes
Results
qPCR Curve
Amplification of cDNA occurred in all six qPCR wells. Melting curve peaks at ~78.5ºC were present in negative controls (C6, D6) and genomic carryover control (E6, F6) wells. Melting curve peaks were at ~81ºC were present in cDNA (A6, B6) and genomic carryover control wells. Melting curve peaks were present at ~86ºC in all wells.
Conclusions
The results of the qPCR procedure are difficult to interpret given the number of peaks. It is evident that contamination or occurred during the procedure and could be due to cross contamination, although micropipettor tips were changed during every transfer of qPCR reactants in an effort to prevent cross contamination. Other explanations such as primer dimer formation or simple genomic carryover do not make sense based on the log fluorescence curve or the melting curve for the reactions. Genomic carryover could result in multiple peaks in the genomic control (E6, F6) and cDNA (A6, B6) wells, but wouldn't result in the peaks in the negative controls (C6, D6). Primer dimer formation would result in the same peak with the same approximate fluorescence intensity across all wells. Therefore, the most viable explanation is likely cross contamination of the qPCR reactants.
Next Steps
Project exploring the expression of the trehalase gene, an enzyme which catalyzes trehalose hydrolysis, during Artemia cyst reanimation and maturation.
10/10/09: Preparation of Artemia hatchery.
11/12/09, 1600: NON-LAB DAY. Addition of Artemia to hatchery and sampling of cysts in duplicate, store in freezer.
11/13/09, 1030: NON-LAB DAY. Collection of Artemia nauplii samples in duplicate from hatchery, store in freezer.
11/16/09, 1030: NON-LAB DAY. Collection of Artemia samples in duplicate from hatchery, store in freezer.
11/17/09: Collection of Artemia sample in duplicate from hatchery, RNA isolation, DNAsing of all samples with TriReagent. Preparation of agarose gel for PCR in subsequent week.-
11/24/09: Reverse transcription of each sample RNA to create template cDNA. PCR of cDNA samples.
Week of 11/31/09: qPCR of cDNA samples.
11/31/09: Electrophoresis of PCR product to test for presence of band expected for trehalase expression.
12/7/09: Project presentation.
27-28 October 2009
Summary
Agarose gel electrophoresis of PCR product, western immunoblot to detect hsp70 protein in Argopecten spp. adductor muscle tissue.
Procedure
PCR Product Electrophoresis
1. Placed pre-stained agarose gel (gel #2) prepared on 10-20-2009 in gel electrophoresis box and removed combs.
2. Covered gel with 1x TAE buffer.
3. Loaded 7µL 100bp ladder in well #1 of the bottom row of wells.
4. Loaded in bottom row of gel #2:
- 25µL of Tube B (PCR Reaction Product) in well #2
- 25µL of Tube C (PCR Reaction Product) in well #3
- 25µL of Tube D (Negative Control) in well #4
- 25µL of Tube E (Negative Control) in well #5
5. Ran gel at 100V for 1 hour.
6. Observed bands using UV transilluminator.
Western Immunoblot Protein Transfer and Visualization
1. SDS-PAGE product gel was prepared in a "sandwich" on semi-dry blotting apparatus in the following order (bottom to top):
- Anode
- Transfer Buffer-soaked filter paper
- Nitrocellulose Membrane
- SDS-PAGE gel
- Transfer Buffer-soaked filter paper
- Cathode
2. Wells were cut off gels
3. Blot "sandwich" was run at 20V for 30 minutes
4. Gel was removed from sandwich and rinsed with transfer buffer
5. Blocking solution was prepared for the gel:
-14ml Ultrafiltered H2O
- 4ml WesternBreeze Blocker A
- 2ml WesternBreeze Blocker B
6. Incubated nitrocellulose membrane on a rocker in blocking solution for 30 minutes.
7. Decanted blocking solution.
8. Rinsed membrane for five minutes, twice, with 20ml H2O.
9. Antibody Solution was prepared:
- 10ml blocking solution
- 3.3µl HSP70 Antibody
10. Incubated nitrocellulose membrane with Antibody Solution for ~18 hours.
11. Rinsed nitrocellulose membrane for five minutes, three times, with 20ml antibody wash.
12. Rinsed nitrocellulose membrane for two minutes, twice, with 20ml ultrafiltered H2O.
13. Incubated nitrocellulose membrane for ~1 hour in WesternBreeze Chromagenic Kit (BCIP & NBT).
14. Rinsed nitrocellulose membrane for two minutes, twice, with 20ml ultrafiltered H2O.
15. Air-dried nitrocellulose membrane.
Results
PCR Product Electrophoresis
A band with weight of approximately ~400-450bp was visible in both PCR reaction lanes (#2, #3), which is likely indicative of a successful PCR since the expected length of the PCR product was 449bp. Faint bands were also visible at ~700bp in the PCR lanes (#2, #3) indicating possible genomic contamination. Bands were visible in all lanes (#2-4) at less than 100bp, indicative of primer dimer formation. (Gel #2 Image )
Western Immunoblot
There were no visible bands in lane #5 (membrane #1), indicating that hsp70 was not present in the Argopecten spp. adductor muscle tissue. (Membrane #1 Image )
Conclusions
Despite the likely genomic contamination and primer dimer formation, the bright band visible at ~400-450bp indicates the presence of tropomyosin mRNA expression in the Argopecten spp. adductor muscle tissue.
The absence of hsp70 protein the Argopecten spp. adductor muscle tissue was expected since the sample specimen was not exposed to stressors which would have triggered expression of any HSF complex genes or the dissociation of the HSF protein complex into active isoforms.
Next Steps
Quantitative PCR to determine the level of expression of tropomyosin mRNA in Argopecten spp. adductor muscle tissue.
20 October 2009
-
Summary
Agarose gel preparation; Argopecten spp. adductor muscle RNA quantification, reverse transcription and PCR.
Procedures
RNA Quantification (continuation from RNA Isolation on 10/13/2009)
1. Thawed sample at room temperature for 5 minutes.
2. Zeroed Nanodrop with 2µL 0.1% DEPC-H2O.
3. Pipetted 2µL sample on Nandrop pedestal.
4. Measured absorbance and concentration at 230nm, 260nm and 280nm.
5. Stored RNA sample at -80ºC.
Agarose Gel Preparation
1. Mixed 2g agarose with 150mL 1x TAE in a 500mL flask.
2. Microwaved solution for 2:15.
3. Swirled to mix and prevent boiling over.
4. Microwaved additional 45 seconds.
5. Cooled solution for ~5 minutes.
6. Mixed 12 µL ethidium bromide into solution.
7. Poured solution into gel tray.
8. Added gel combs and allowed gel to cool and set.
9. Placed gel in refrigerator for storage.
Reverse Transcription and PCR
1. Mixed RNA sample by inverting tube.
2. Added 5µL sample to clean PCR tube (Tube A).
3. Incubated tube in thermal cycler 75ºC for 5 minutes
4. Transferred tube to ice and allowed sample to cool for 5 minutes.
5. Added to sample tube:
-4µL 5x AMV RT Buffer
-8µL dNTPs
-1µL Oligo dT Primer
-1µL AMV RTranscriptase
-1µL RNase free H2O
6. Vortexed and spot-spinned to mix.
7. Incubated at room temperature for 10 minutes.
8. Incubated for 1hr at 37ºC in thermocycler to transcribe cDNA.
9. Prepared primers (repeated for each of forward and reverse primers):
a. Centrifuged dehydrated primers
b. Added 10µL of DNAse/RNAse free H2O per nM dehydrated primer.
c. Transferred 100µL of 10µM primer solution to clean 1.5µL RNAse/DNAse free snap cap tubes.
10. Prepared PCR master mix for 50µL reaction volumes combined and mixed in 1.5µL DNAse/RNAse free snap cap tubes:
- 125µL GoTaq Green Master Mix, 2X
- 12.5µL 10µM upstream primer
- 12.5µL 10µM downstream primer
- 90µL DNAse/RNAse free H2O
11. Added 48µL of PCR master mix to tubes B, C, D and E.
12. Deactivated transcription (Tube A) by heating for 3 min at 95ºC in thermocycler.
13. Spot spinned template cDNA sample (Tube A) for ~5 seconds.
14. Transferred template cDNA (Tube A) to ice briefly (~1 min) to cool.
15. Added 2µL template cDNA to tubes B and C.
16. Added 2µL DNAse/RNAse free H2O to tubes D and E (negative controls).
17. Tube A and primers stored at -80ºC.
18. Tubes B-E placed in thermocycler for cDNA denaturation at 95ºC for 10 minutes.
19. Forty (40) amplification cycles for tube B-E:
a. 95ºC - 30 sec (Denaturation)
b. 55ºC - 30 sec (Annealing)
c. 72ºC - 90 sec (Extension)
20. Final extension at 72ºC for 3 minutes.
21. PCR products in tubes B-E stored at 4ºC.
Results
RNA Quantification
RNA Concentration: 450.3 ng/µL
A260 Absorbance: 11.257
A260/A280 Ratio: 1.86 (Within range for clean RNA)
A260/A320 Ratio: 0.86 (Outside range for clean RNA)
Reverse Transcription and PCR
Primer Preparation
Forward Primer Sequence for Argopecten spp. tropomyosin: 5'-CCTGGCCGAACAGATGGAGCA-3'
Reverse Primer Sequence for Argopecten spp. tropomyosin: 5'-CAAGCTTACGGGCGGCCTCA-3'
Dehydrated Forward Primer: 31.0 nM * 10µL DNAse/RNAse Free H2O = 310 µL 10µM Forward Primer
Dehydrated Reverse Primer: 33.3 nM *10µL DNAse/RNAse Free H2O = 333 µL 10µM Reverse Primer
PCR Tube Contents
Tube A - Template cDNA
Tube B - PCR Reaction #1 (48µL PCR Master Mix + 2 µL Template cDNA)
Tube C - PCR Reaction #2 (48µL PCR Master Mix + 2 µL Template cDNA)
Tube D - Negative Control (48µL PCR Master Mix + 2µL DNAse/RNAse free H2O)
Tube E - Negative Control (48µL PCR Master Mix + 2µL DNAse/RNAse free H2O)
Conclusions
The sample Argopecten spp. adductor muscle RNA quantification indicated an unclean sample based on the A260/A230 ratio. The determined ratio of 0.86 was lower than the ratio for a clean sample (1.5-2.0), indicating the presence of ethanol, phenol or salts in the sample. This was likely due to the carryover of interphase debris during RNA isolation (Step #11, 10/13/09) since I tried to remove as much of the aqueous phase as possible. It also could have occured during EtOH removal (Step #18, 10/13/09), but the large size of the pellet prior to pipetting off EtOH (noted on 10/13/09) was likely indicative of contaminant carryover from a previous step. Regardless of the cause, the low A260/A230 ratio was indicative of an unclean sample, making the measured RNA concentration of 450.3 ng/µL unreliable.
If the RNA sample is clean, the amount of RNA used during reverse transcription (2.25µg) would exceed the recommended amount for reverse transcription (0.25µg). Due to the contamination, it is likely that the final PCR product will be unreliable.
Next Steps
Run the PCR end-product out on ethidium bromide-stained agarose gel to test for tropomyosin expression in Argopecten spp. adductor muscle. Expected 449bp product from positive samples B, C. No product expected from negative controls D, E.
13 October 2009
Summary
Argopecten spp. adductor muscle RNA isolation and SDS-PAGE.
Procedures
RNA Isolation (continued from week 1)
6. RNA sample thawed at room temperature.
7. 200µL chloroform added to sample tube.
8. Vortexed sample for ~30s.
9. Incubated at room temperature for ~5 minutes.
10. Microfuged in refrigerator at maximum speed for ~15 minutes.
11. Transferred aqueous phase to fresh RNAase free 1.5µL sample tube.
12. Added 500µL isopropanol to sample tube.
13. Mixed by inversion and incubated at room temperature for ~10 minutes.
14. Microfuged in refrigerator at maximum speed for 8 minutes.
15. Supernatant removed from sample.
16. Added 1mL 75% EtOH to sample tube, vortexed to loosen pellet.
17. Microfuged in refrigerator at 7500g for 5 minutes.
18. Removed supernatant.
19. Microfuged for an additional 15 seconds.
20. Removed remaining supernatant, and allowed pellet to dry for ~3 minutes.
21. Added 100µL 0.1% DEPC-H2O to sample tube.
22. Dissolved pellet by mixing with pipette.
23. Incubated in 55ºC water bath for ~5min.
24. Flicked tube to mix.
25. Stored at 80ºC, quantification to be performed next week.
Protein Gel Protocol (SDS-PAGE)
1. Thawed protein sample.
2. Mixed sample by inversion in 1.5mL snap cap tube.
3. Combined 15µL sample and 15µL 2x Reducing Sample Buffer in 1.5µL screw cap tube.
4. Flicked tube to mix and centrifuged for ~10 seconds.
5. Boiled sample in water bath for 5 minutes.
6. Centrifuged sample tube for ~1 minute.
7. Loaded sample into SeeBluePlus2 gel*, but couldn't load entire sample. Only added ~11µL of sample (9.57µg protein) into well #9.
8. Ran gel for ~35 minutes at 150 volts.
9. Removed gel from gel box, placed in tray.
10. Coomassie Stain was added to cover gel in tray.
11. Gel tray incubated on rocker for 5 minutes.
12. Stain was poured poured out of tray.
13. Gel was rinsed briefly with 10% acetic acid solution.
14. 10% acetic acid solution added to tray.
15. Gel tray incubated incubated on rocker for ~15 minutes.
16. Changed acetic acid solution out, incubated for an additional ~15 minutes.
*Gel box filled with 4-20% Tris-Hepes buffer SDS.
Results
SDS-PAGE
Gel Image
Only 11µL (not 30µL) of sample was added to the well in the gel due to an air bubble in the tip of the micropipette. The Argopecten spp. adductor tissue SDS-Page (lane #9) showed bands at ~28kDa, ~38kDa, ~98kDa and ~188kDa.
RNA Isolation
Pellet was present after step #21.
Conclusions
SDS-PAGE
Since there was not enough sample added to the gel well, it's likely that protein present in lower concentrations in the sample tissue were not sufficient to generate visible bands upon completion of the SDS-PAGE.
RNA Isolation
The presence of a sizeable pellet after step #21 is a likely indicator that RNA is indeed present in the sample to be quantified next week using a Nanodrop protocol.
Next Steps
1. Complete RNA quantification using Nanodrop protocol.
2. Reverse transcription and PCR to test for the presence of tropomyosin in Argopecten spp. adductor muscle tissue.
6 October 2009
Summary
- Homogenization and lysing of tissue (using TriReagent) performed on bay scallop for RNA extraction.
- Protein extraction and quantification performed on bay scallop tissue using CelLytic MT solution and Bradford Assay procedures.
Procedures
RNA Isolation
1. Combined 500µL TriReagent with 50-100mg frozen bay scallop (Argopecten spp.) tissue in 1.5 mL snap cap tube.
2. Homogenized tissue in tube.
3. Added 500µL additional TriReagent to snap cap tube.
4. Vortexed for 15 seconds.
5. Stored sample at -80ºC.
Protein Extraction and Quantification
1. Combined 0.5mL CelLytic MT solution with 25mg bay scallop (Argopecten spp.) tissue in 1.5mL snap cap tube.
2. Homogenized tissue in tube.
3. Mixed tube contents by inverting several times.
4. Microfuged (refrigerated) for 10min at maximum speed.
5. Transferred supernatant to new tube, stored on ice.
6. Diluted 15µL sample with 15µL deionized H2O in new 1.5mL snap cap tube (sample).
7. 30µL deionized H2O added to a new snap cap tube (blank).
8. 1.5mL Bradford reagent added to each tube.
9. 1mL blank and 1mL sample added to disposable cuvettes.
10. Blank used to zero spectrophotometer.
11. Sample absorbance measured twice at 595nm. Mixed sample in cuvette between spectrophotometric measurements.
12. Protein concentration back-calculated using standard curve for Bradford protein assay.
13. Sample stored at -20ºC.
Results
Protein Quantification
Absorbance 1: 0.436
Absorbance 2: 0.424
Absorbance Mean (x): 0.430
Dilution Factor: 2
Bradford Curve Equation: 1011.9x
Bay Scallop Tissue Protein Concentration = 1011.9(0.430) * 2 = 870.234µg/mL
Conclusions/Next Steps
The protein extraction procedure was successful in quantifying the protein concentration of bay scallop tissue.
The next step will be to continue with RNA isolation and quantification procedures and run a SDS polyacrylamide gel electorophoresis (SDS-PAGE) on the protein sample.