Cloning of DD products
PCR of restreaked colonies
Master Mix (x35)
2xApex buffer 875 uL
water 805 uL
Pf 35 uL
Pr 35 uL
Aliquoted 50 uL of MM into plate wells. Touched sterile plastic wand to restreaked colonies and set in MM. Swirled wands in MM before removing. Program CLNY in SBR directory.
Made 1.2% agarose gel and ran 20 uL of colony PCR product at 100V for ~30 minutes.
Based on gel band sizes, did Qiagen minipreps for sequencing of the following samples (PS#-colony#): 10-4, 10-5, 10-6, 12a-1, 12a-3, 12a-5, 9-1, 9-4, 10-4, 10-5, 10-6, 12b-1, 12b-4, 18-1, 18-3, 38-2.
Followed Qiagen MinPrep protocol to extract DNA from chosen cultured colonies.
Poured ~1.5 mL of each LB+colony into minicentrifuge tubes. Spun at max speed, 2 min. Discarded supernatant and poured in 1.5 mL more, spun, and decanted (white pellet on bottom).
Resuspended pelleted bacteria in 250 uL Buffer P1 via vortexgin.
Added 250 uL Buffer P2 and mixed by inverting 4 times.
Added 350 uL Buffer N3 and immediately mixed by inverting 4 times.
Transferred supernatant to QIAprep spin column and centrifuged at 13000xg for 1 min. Washed column with 750 uL Buffer PE and spun at max speed for 1 min. Discarded flow through and spun additional minute at max speed.
Put column in new eppie tube, added 30 uL Buffer EB. Let incubate at RT for 1 minute, then spun for 1 min.
Put 10 uL of eluted DNA into plate to send off for sequencing.
Results: Successful cloning of gene products from second band in primer set 12 and primer sets 9, 18, and 38.
December 2, 2009
Cloning of DD products
Left plates from 12.1.09 to incubate longer at 37 C to allow for more colony growth.
Grid a plate with 8 squares for PS#10 colonies and 8 squares for PS#12 (200 bp) and 2 squares for a negative control (dark blue colony) from each.
Prepped master mix:
| Reagent |
volx1 |
volx18 |
| 2xApex buffer |
25 uL |
450 uL |
| water |
23 uL |
414 uL |
| Pf |
1 uL |
18 uL |
| Pr |
1 uL |
18 uL |
Using sterile plastic wand, picked colonies, streaked them in the appropriate plate grid, and then placed wand(tip first) in corresponding MM-filled well in plate.
| Well |
Sample |
vol/color |
Well |
Sample |
vol/color |
| A1 |
10-1 |
50/W |
A2 |
12-1 |
50/W |
| B1 |
10-2 |
50/W |
B2 |
12-2 |
50/B |
| C1 |
10-3 |
50/W |
C2 |
12-3 |
50/W |
| D1 |
10-4 |
50/W |
D2 |
12-4 |
100/W |
| E1 |
10-5 |
100/W |
E2 |
12-5 |
100/B |
| F1 |
10-6 |
100/W |
F2 |
12-6 |
100/B |
| G1 |
10-7 |
100/W |
G2 |
12-7 |
100/B |
| H1 |
10-8 |
100/W |
H2 |
12-8 |
100/W |
Thermal profile:
1. 94C, 8 minutes
2. 94C, 45s
3. 50C, 1 min
4. 72C, 1 min, 30s
rep 2-4 39 more times
5. 72C, 10 min
Made 1.2% agarose gel (1.2 g agar, 100 mL 1xTAE, 10 uL EtBr). Loaded 20 uL 100 bp ladder and PCR product. Ran at 100 V for ~45 minutes.
Gel 1:
ladder A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 F2 G2
Gel 2:
ladder H2 A3 B3
Restreaked and seeded all colonies grown on 12.1.09 (see table below). Made 200 mL LB+Kan: 50 mL 5x lab stock + 160 mL nanopure H2O + 200 uL Kan. Aliquoted 5 mL of LB mix. to glass tubes with sponge stoppers. Touched a sterilized toothpick to each colony and dropped into growth medium. Based on gel results above, did the same for chosen colonies of PS#10 and PS#12a that appeared to have different products: 10-4, 10-5, 10-6, 12a-1, 12a-3, 12a-5. Grew up overnight at 37C, 250 rpm. Did not exceed 8 colonies for any primer pair.
Results: Successful cloning of gene products with primer sets 10 and 12. Possibly 3 different products per primer set (to be determined by sequencing).
December 1, 2009
Cloning of DD products
Repeated procedure from 11.30.09 for primer sets 1, 9, 12 (400 bp band), 18, 38, and 57
The following changes were made to the procedure:
cloning reaction was cut in half so only 2 uL PCR product, 0.5 uL salt, and 0.5 uL vector were used.
LB+Kan plates were streaked with volumes of either 50 uL or 200 uL competent, transformed cells.
Plates were streaked around 3 pm and left to incubate overnight at 37 C.
Results: Around 7 am 12.2.09 very few and faint colonies were formed (some white, some blue).
November 30, 2009
Cloning of DD products
Used TOPO TA cloning kit (Invitrogen), T10 competent cells.
PCR products to clone: primer set #10 (11.19.09), PS# 12 (11.17.09) 200 bp band.
Let bands thaw at room temp. Spin at 5000xg for 10 min (RT) in ultrafree-DA tubes.
For cloning reaction in strip tubes:
4 uL PCR product
1 uL salt solution (TOPO kit)
0.9 uL vector (TOPO)
Put on thermocycler for 10 min at 22C, then directly to ice.
Added 2 uL of each cloning reaction to tube of competent cells (thawed on ice right before use); swirled as added. Icubated on ice for 10 minutes, then heat shock in 42C water bath for 30 s. Put on ice for 2 minutes.
Added 250 uL room temp SOC medium to competent cells+cloning reaction. Rolled tubes to coat sides.
Put in 37C incubator at 225 rpm for 1 hour.
Warmed LB +Kan plates to RT (4 plates total, 2 for each primer set). Spread with 80 uL 20mg/mL Xgal. Dried plates at 37C.
Plated out transformed cells, for each primer set plated 1 plate with 50 uL and one with 100 uL. Incubated at 37C with lids cracked to dry residual liquid and then upside down overnight.
Results: Blue and white colonies appeared on all 4 plates. Colonies picked and restreaked 12.2.09.
November 25, 2009
qPCR
qPCR in duplicate of EF1, HIF , MDR, and Prx6 . Used cDNA from 11.6.09
Results: Amplification of samples, no contamination in blanks. Possible differential expression of CO2 vs control, but need to do stats to make sure.
November 24, 2009
Differential Display
Loaded PCR products from 11.23.09 according to gel layout. Loaded 7 uL of each PCR product and 20 uL of 100 bp ladder on 2% agarose gel. Ran at 100 V for ~45 minutes. Imaged with UV.
Results: Differential expression (high MW) in CO2 pool at primer set 57. Cut out band and stored at -20C
Cloning of DD Products
Diluted 100 mL of 5xLB (lab stock) with 400 mL nanopure water in large flask. Added 7.5 g Bacto Agar. Put in autoclave (121C, 20 minutes). After autoclaving, let LB cool (in 50C water bath) until flask is comfortable to touch for at least 30s. Added 500 uL Kanamycin for 50 ug/mL. Swirled flask until homogenated. Poured LB + kan into sterile petri dishes with lids so that LB covered bottom. Let sit undisturbed (~30 minutes) until set. SW packaged and put in fridge.
November 23, 2009
Differential Display
Gene fishing (SeeGene) PCR with 3 new primer sets: 56, 57, and 58.
Plate layout and gel layout:
November 22, 2009
Project: Differential gene expression of CO2-exposed juvenile Pacific Oysters
The following procedures are what I have accomplished up to this point for the final laboratory project. The work flow is organized to linearly follow the procedures, even though the work was not always performed in such a linear fashion. Dates are indicated where results were obtained.
I. Oyster CO2 Challenge and Sample Collection
Adult and juvenile Pacific oysters (Crassostrea gigas) were exposed to elevated pCO2 (970 ppm) or present-day levels of CO2 (350 ppm). These groups will be referred to as “CO2” and “control”, respectively. Directly after CO2 exposure, 8 CO2 and 8 control juveniles were sacrificed and whole body samples were stored at -80C. The following procedures were performed on these 16 samples.
II. RNA Extraction (Oct. 7 and Oct. 14, 2009)
The first round of RNA extractions were performed on 4 of the 16 samples, CO2-5, CO2-6, control 7, and control 8. Samples weights and corresponding ½ volumes of Tri Reagent were as follows:
| Sample |
Weight(g) |
1/2 Vol. Tri (mL) |
| CO2-5 |
0.17 |
1 |
| CO2-6 |
0.38 |
2 |
| Cont7 |
0.16 |
1 |
| Cont8 |
0.86 |
4.5 |
Followed manufacturer’s protocol for remaining RNA extraction. During first spin (10000 rpm for 15 minutes at 4C), CO2-5 and control 7 tubes shattered in rotor tubes. Removed samples from rotor, put in new falcon tubes and respun. Continued protocol. RNA was resuspended in 50 uL nuclease-free H2O and stored at -80C.
After RNA extraction completed, measured concentration on Nanodrop.
| Sample ID |
ng/uL |
A260 |
A280 |
260/280 |
| CO2-5 |
294.4 |
7.36 |
3.7 |
1.99 |
| CO2-6 |
565.61 |
14.14 |
7.212 |
2.34 |
| Cont7 |
650.46 |
16.26 |
7.893 |
2.06 |
| Cont8 |
2194.82 |
54.87 |
27.557 |
1.95 |
| Sample |
Weight(g) |
1/2 TriReagent vol. (mL) |
| CO2-1 |
0.29 |
1.5 |
| CO2-2 |
0.28 |
1.5 |
| CO2-3 |
0.56 |
3 |
| CO2-4 |
0.22 |
1.25 |
| CO2-7 |
0.53 |
2.75 |
| CO2-8 |
0.2 |
1 |
| Cont1 |
0.37 |
2 |
| Cont2 |
0.33 |
1.75 |
| Cont3 |
0.38 |
2 |
| Cont4 |
0.48 |
2.5 |
| Cont5 |
0.37 |
2 |
| Cont6 |
0.88 |
4.5 |
III. Real-time Quantitative PCR of RNA to determine Genomic Contamination
Each PCR included: oyster RNA, 4 negative controls (water), 1 negative control of just master mix, and 3 positive controls of genomic C. gigas DNA at dilutions 1:10, 1:100, 1:1000. PCR was done with 18s C. gigas primers.
Master mix preparation and PCR conditions outlined in data sheets.
Oct 12, 2009 -
Oct 15, 2009
Results: evidence of gDNA contamination in all RNA samples. Will need to enzymatically remove DNA from RNA (gDNA clean-up).
IV. Genomic DNA Clean-up
For each RNA sample, added either 10 or 5 uL RNA to 40 or 45 uL water. Amount was determined based on concentrations from Nanodrop. Samples with concentrations >1 ug/mL were diluted 5 uL RNA in 45 uL water; samples with concentrations <1 ug/mL were diluted 10 uL RNA in 40 uL water. The 10 uL samples were CO2-1, CO2-5, CO2-6, and cont7.
To the 50 uL of diluted RNA, added 5 uL 10x TURBO Buffer (Ambion TURBO DNA free kit) and 2 uL TURBO DNase (2U). Mixed gently and spun down.
Incubated samples in 37C water bath for 30 minutes. Added 5 uL DNase Inactivation Reagent to each tube (before adding, vortexed DIR to resuspend).
Vortexed samples with DIR. Incubated at room temperature for 2 minutes, vortexign twice during incubation.
Centrifuged samples at 10000xg for 1.5 minutes. Transferred RNA (supernatant) to clean tubes labeled with name of sample, “RNA”, “gDNA-free”, date, and initials. After PCR< stored cleaned RNA at -80.
Repeated qPCR with 18S C. gigas primers to check for successful removal of gDNA.
Results: Samples all amplified, which indicates gDNA contamination. Suspected that had not diluted RNA enough (to accurately represent what will be final cDNA concentration), so repeated qPCR with 1:20 dilutions RNA. Still evidence of gDNA contamination, however.
Repeated DNase protocol on previously-DNased RNA samples (these are now 2xDNased). Diluted samples 1:20 for qPCR contamination check. Repeated qPCR with 18s primers.
Results: No amplification in experimental samples, but appropriate amplification in positive controls. Negative controls were also clean.
V. Reverse Transcription: RNA to cDNA
Reverse transcribed RNA (2x DNased samples) to cDNA. Transferred 5 uL of each sample to a strip tube well. Incubated at 75C for 5 minutes. Transferred to ice for 5+ minutes.
Prepared master mix (x18):
72 uL 5x AMV RT buffer
144 uL 10 mM dNTP mix
18 uL AMV RTranscriptase
18 uL Oligo dT primer
18 uL RNase free water
Aliquoted 15 uL of master mix into each sample well. Incubated at room temperature for 10 minutes. Incubated at 37C for 1 hour then heat deactivation at 95 degrees for 3 minutes. Stored at -20C.
VI. Real-time Quantitative PCR of Oyster cDNA
Oct 29, 2009 : elongation factor 1 (EF1), interleukin 17 isoform D (IL-17), cytochrome P450 (cytp450)
Nov 2, 2009 : multi-drug resistance (MDR), superoxide dismutase (SOD), and AURKA
Nov 3, 2009 : methallothionein IV (MTIV), heat shock protein 70 (hsp70), peroxiredoxin 6 (prx6), hypoxia-induced factor 1a (HIF1a)
Results: used EF1 to normalize expression of other genes. Possible differential expression between CO2 and control, but need replicated PCRs to determine verity.
VII. Reverse Transcription II
Used a different RT protocol (MMLV). For a 25 uL reaction, approximately 1 ug of total RNA per reaction.
Added 1 uL from each sample of CO2 challenged )n=8) and control (n=8) to a well in a PCR plate. To each sample added: 16.75 uL nuclease-free water and 0.5 uL Oligo dT primers. Heated samples at 70C for 5 minutes. Transferred immediately to ice for <10 minutes.
Prepared master mix for 17 reactions:
| Reagent |
Volx1(uL) |
Volx17(uL) |
| 5x MMLV Buffer |
5 |
85 |
| 10 mM dNTPs |
1.25 |
21.25 |
| MMLV RTranscriptase |
0.5 |
8.5 |
| RNA template + Oligo dT primer |
VIII. Quantitative PCR of Oyster cDNA
Diluted new cDNA 1:10 by putting entire cDNA in 225 uL of water (in 1.5 mL eppie tubes, stored at -20C). Prepared standard master mix and did qPCR of EF1, Prx6, HIF, and MDR in duplicate.
qPCR Nov 6, 2009 (EF1 & Prx6)
qPCR Nov. 9, 2009 (HIF & MDR)
IX. Differential Display
A different approach to finding differential gene expression with no a priori decisions as to which genes are involved in the physiological response to the treatment.
A. Pool RNA samples to make one pool of all treatment and one pool of all control.
Nanodropped RNA samples (from the 2x DNased). Measured concentrations 3 times and averaged to find the final concentration. Based subsequent calculations on this final value.
Calculated amounts of each (of 8) RNA samples that would go into the pool so that all were represented equally (in terms of amount). Pool 1 is the CO2 challenged oysters and Pool 2 is the control group.
B. To get correct amount of RNA, need to precipitate because volumes are too large. To each pool, added 0.1 volume 5M ammonium acetate (9.36 uL for Pool 1 and 7.63 uL for Pool 2). Added 2 volumes 100% EtOH (205.9 uL and 167.88 uL, respectively) to each pool. Mixed well and incubated at -80C for 30 minutes.
Spin tube at max speed at 4C for 30 minutes.
Discarded supernatant and did quick spin for removal of residual EtOH.
Added 1 mL 70% EtOH; flicked tube to get pellet off bottom.
Spin at max speed, 4C for 10 minutes. Discarded supernatant. Removed residual EtOH.
C. Resuspended pooled RNA in 18 uL water. Heated at 55C for 5 minutes. Measured concentrations (ng/uL) of pools on Nanodrop.
| Pool 1 |
Pool 2 |
| 169 |
26 |
| 170.3 |
|
| 172.3 |
Repeated pooling and precipitation of Pool 2. Resuspended in 9.5 uL DEPC H2O. New concentrations:
| Pool 2 (ng/uL) |
| 737.9 |
| 745.4 |
| Avg: 741.65 |
Reverse transcription reaction for Pool 1: 7.5 uL RNA + 2 uL 10 uM dT-ACP1
For Pool 2: 1.75 uL RNA + 2 uL 10 uM dT-ACP1 + 5.75 uL water
Prepared RT reactions as outlined above. Incubated tubes at 80C, 3 minutes then to ice for 2 minutes. Added to each tube: 4 uL RT 5x MMLV buffer, 4 uL 2.5 mM dNTPs, 1.5 uL water, 1 uL reverse transcriptase (MMLV). Incubated on thermocycler: 42 C 90 minutes, 94C 2 minutes. Chilled for 2 minutes on ice then added 80 uL water to each tube. Vortexed to mix.
E. PCR of pooled cDNA with arbitrary primers
Amplified cDNA with arbitrary primers to find differential expression. For each primer set, did PCR for CO2 and control groups. PCR reactions were as follows:
3 uL cDNA
2 uL 5 mM arbitrary primer
1uL 10 uM dT-ACP2
4 uL distilled water
10 uL SeeAmp 2x master mix
Thermal profile for PCR:
Before PCR pre-heat block to 94C.
- 94C, 5 min
- 50C, 3 min
- 72C, 1 min
- 40 cycles of: 94C, 40s; 65C 40s; 72C 40s
PCR was performed with the following primer sets from the SeeGene kit: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 33, 34, 35, 37, 38, 39, 40.
When PCR finished, made 2% agarose gel with ethidium bromide (2 g agar, 100 mL 1xTAE, 10 uL EtBr). Loaded 20 uL of 100 bp ladder on each gel run and 7 uL of each sample (control loaded next to CO2 for each primer set).
Results:
Gel 11.13.09
Gel 11.17.09
Gel 11.19.09
Gel 11.20.09
Differential display detected with following primers – each primer number is followed in parentheses by the group that showed greater expression
November 3, 2009
Lab 5: Quantitative PCR
Prepared Master mix, one for each primer pair. Prepped a mix for 7 samples to perform PCR for each primer pair on 2 replicates of Vibrio cDNA, 2 replicates of a negative control (water), and 2 replicates of RNA to check for genomic DNA contamination. (All volumes are in microliters)
Reagent Volx1 Volx7
Immomix 2x 25 175
Syto13 dye 2 14
10 uM Pf 1 7
10 uM Pr 1 7
H2O 19 133
Combined above ingredients in 2 separate tubes and mixed well. Aliquoted 48 uL of master mix into each well of strip tube for PCR, one strip tube for each primer pair. Then added 2 uL water, cDNA, or diluted RNA (1:4) in each well (layout below). "1" designates primer pair 1, likewise for "2". Closed strip tubes and gave to MG to load qPCR.
| Neg1 |
Neg1 |
cDNA1 |
cDNA1 |
RNA1 |
RNA1 |
RNA2 |
RNA2 |
cDNA2 |
cDNA2 |
Neg2 |
Neg2 |
Real time qPCR melting curve here . The negative controls are all clean (they did not amplify), however there is genomic DNA carry-over. The RNA and cDNA samples amplified similarly, indicating that there is double-stranded DNA in the RNA. There is only one melting point, however, indicating that only one PCR product was amplified in both cDNA and RNA (these results are true for both primer pairs). If I were to continue with these samples, I would need to clean the RNA with DNase in order to proceed with analysis.
Next Steps...
Planned Project Schedule:
Already accomplished: Experimental treatment (CO2 exposure), RNA extraction, gDNA clean-up (DNase), reverse transcription to cDNA, initial qPCR.
11/10: qPCR to replicate previous results. Order primers for potential genes of interest found in literature.
materials: reagents for qPCR
11/17: Analyze results to date. qPCR with new primers.
materials: reagents for qPCR
11/24: Extract RNA from Vibrio-exposed juveniles. Quantify on Nanodrop and PCR to check for genomic contamination.
materials: RNA extraction reagents, qPCR reagents and 18s primers.
12/1: Reverse transcribe new juvenile oyster RNA. qPCR for genes of interest.
materials: RT reagents, qPCR reagents.
12/8: Analysis and write-up
October 27, 2009
-
Lab 4: Western Transfer-Immunoblots
I: Run PCR Products on Gel
Put gel from last week in gel box and filled until gel was covered with 1x TAE buffer. Removed combs. Loaded 7 uL 100 bp ladder in the leftmost wells of 1st and 2nd rows. Loaded 25 uL samples in first row after ladder in following order: blank for primer set 1, blank 1, sample for primer set 1, sample 1, blank 2, blank 2, sample 2, sample 2. ED loaded in 2nd row.
Ran gel at 100 volts for about 1 hour. Visualizedgel on UV transilluminator.
Results: Expected amplification at ~110 bp for primer set 1 and ~229 for primer set 2 (faint primer dimers visible in blanks).
II. Western Blotting
Pre-lab, MG cooled transfer buffer to 4 degrees C and soaked filter paper, membrane and gel in Transfer buffer for 15 minutes. Made blotting sandwich, smoothing layers as laid them down, in following order: Anode, filter paper, nitrocellulose membrane, protein gel*, filter paper, cathode.
*Not the same gel made in lab Oct. 13. MG remade gel so that protein amounts were consistent across samples. Vt + oyster is on gel 2, lane 3 (after ladder & ED's Vt). 3 ug of sample was loaded.
Results: Proteins of multiple sizes present but at low concentration (which is to be expected since only 3 ug of the sample was loaded).
Transferred blot for 30 minutes at 20 volts. Removed gel from sandwich and rinsed with transfer buffer. Removed adhering gel from membrane. In parallel, took protein gel and stained with Coomassie for 5 minutes on rocker, then rinsed with 10% acetic acid briefly, and followed with progressive 10% acetic acid washes ever 15 minutes on rocker until destained. Visualized on light box.
Prepared 20 mL of Blocking Solution (all reagents from Western Breeze kit) with 14 mL ultra filtered water, 4 mL blocker/diluent (Part A), 2 mL blocker/diluent (Part B). Placed membrane in 10 mL of blocking solution in covered , plastic dish. Incubated for 30 minutes on shaker. Decanted blocking solution. Rinsed membrane with 20 mL of water for 5 minutes and decanted (repeated once).
Prepared 10 mL of primary antibody solution (1:3000 dilution) of 10 mL blocking solution and 3.3 uL HSP 70 antibody. Incubated membrane in primary antibody solution overnight.
Next steps: qPCR of cDNA samples (reverse transcribed from vibrio RNA) to quantify expression of metalloprotease gene.
October 20, 2009
Lab 3: Reverse Transcription & PCR
I: RNA Quantification
RNA was quantified last week (Oct 13, 2009). See end of "PartB: RNA Isolation" for methods and results.
II: Reverse Transcription of RNA to cDNA
Thawed RNA isolated 10/13/09 and mixed by inverting tube a few times. Transferred 5 uL of the RNA to a new 0.2mL tube. Incubated RNA at 75 degrees C for 5 minutes in thermal cycler. Transferred tube to ice and incubated 5 more minutes. To tube containing RNA added reagents for RT PCR:
4uL 5x buffer (AMV RT Buffer)
8 uL dNTPs (10 mM total)
1 uL AMV RTranscriptase
1 uL Promega Random Primers 500 ug/mL
1 uL RNase free water
(total volume = 15 uL)
This reaction will make double-stranded cDNA from single-stranded RNA.
Vortexed tube, spot spin, and incubated at room T for 10 minutes. Put in thermocycler at 37 degrees C for 1 hour. After 1 hour, heat inactivation of enzyme at 95 degrees for 3 minutes. Spot spin tube and put on ice. This will be the template for the next PCR reaction.
III: PCR with designed primers (Vt metalloprotease 1 & 2)
At beginning of lab, the 2 primer pairs (4 primers) were reconstituted with nuclease-free H2O. The primers were designed from published sequence of Vibrio tubiashii metalloprotease A (the 2 sets of primers amplify non-overlapping segments of the published sequence). Primer set 1 should have a 110 bp amplicon and primer set 2 should have a 220 bp amplicon. The primers were reconstituted in the following volumes to achieve 100 uM stocks:
ETS 1 F Vt MTPase 33.4nm --> 334 uL
ETS 1 R Vt MTPase 40.9 nm --> 409 uL
ETS 2 F Vt MTPase 33.6 nm --> 336 uL
ETS 2 R Vt MPTase 39.9 nm --> 399 uL
100 uM stock solutions were then diluted to make 10 uM working stocks: 10 uL of stock primer in 90 uL nuclease-free H2O.
Prepared master mix for PCR of cDNA. Mix was prepared for 5 reactions - the sample in duplicate and 2 negative controls (+ extra for pipetting error). Separate mixes were prepared for each primer pair.
Reagent Volume/rxn Volume*5
GoTaq Green Master Mix, 2x 25 uL 125 uL
upstream primer, 10 uM 2.5 uL 12.5 uL
downstream primer, 10 uM 2.5 uL 12.5 uL
H2O 18 uL 90 uL
cDNA template 2 uL --
2 uL of template (water for negative controls) and 48 uL of master mix were pipetted into 0.2mL reaction tubes. PCR was accomplished according to the following thermal profile:
1. 95 degrees C, 10 min
2. 95 degrees, 30s
3. 55 degrees, 30s
4. 72 degrees, 90 s
(repeat 2-4 40 times)
5. 72 degrees, 3 min
6. 4 degrees forever
IV: Preparation of Agarose Gels for next week
Weighed 2 g of agarose and mixed with 150 mL 1x TAE in 1L flask. Microwaved for ~ 3 minutes. (Boiled over so added a little more TAE and microwaved again briefly until all agar dissolved.) Let cool a few minutes. Added 12 uL ethidium bromide. Mixed thoroughly by swirling. Poured gel into gel tray with 2 combs (large). Got rid of bubbles. After gel set (~20 minutes) wrapped in plastic and stored in fridge.
Next steps: Western transfer immunoblots of the protein samples to further separate and quantify proteins. PCR'd samples will be run on a gel to make sure that correct amplicon has been amplified based on primers designed.
October 13, 2009
Lab 2: Tissue Extraction
PartA: Protein Gel Protocol
Thawed protein extraction from Oct. 6. Mixed well via inversion. In new 1.5mL screw cap tube, mixed (by flicking) 15 uL protein sample and 15 uL 2x Reducing Sample Buffer. Spot centrifuged tube and boiled for 5 minutes in water bath on hot plate. Did another quick spin after boiling.
Tris-Hepes 4-20% (becomes denser from top to bottom) gel was placed in rig and rig was filled with 1x Tris-Hepes buffer SDS. Loaded entire 30 uL of sample into well 2 of gel (3.21 ug of protein). In well 1, Mac had loaded 10 uL of SeeBlue ladder Plus2 (Invitrogen). Molecular weights correspond to NuPAGE MES gel. Ran gel at 150V for 30 minutes. Removed gel from rig and covered with Coomassie Stain. Incubated gel on rocker for 5 minutes. Removed stain and rinse gel with 10% acetic acid, pour wash out when done. Add fresh acetic acid and incubate on rocker for 15 minutes. Changed buffer ever 15 minutes until image of proteins on gel clear. Took picture of gel . Clear bands for Vt+oyster protein at ~150 kDa, 98, ~60, 38, and 28 kDa.
PartB:RNA Isolation
Incubated protein sample at RT for 5 minutes. In fume hood, added 200 uL chloroform. Vortexed vigorously for 30s until sample became milky emulsion. Incubated at RT for 5 minutes. Put tube in refrigerated microcentrifuge for 15 minutes at max speed. Transferred top, clear, aqueous phase to fresh eppie tube. Disposed remaining pink waste in phenol/chloroform receptacle in hood. Added 500 uL isopropanol to RNA tube and mixed by inversion numerous times. Incubated at RT for 10 minutes then put in refrigerated centrifuge at max speed for 8 minutes. Pellet was not clearly visible, but knew where in tube it should be (based on orientation in centrifuge), so pretended it was there. Removed supernatant with pipette. Added 1 mL of 75% EtOH to "pellet" and vortexed briefly. Put in centrifuge for 5 min at 7500 g. Removed supernatant carefully to not dislodge invisible pellet, spot centrifuge, and removed remaining EtOH. Let pellet dry at RT (open tube) for 5 minutes. Resuspended pellet in 100 uL DEPC H2O, pipetting a few times to dissolve and heated at 55 degrees C (water bath) for 5 minutes. Removed tube from heat, flicked and put on ice. Quatitated RNA on Nanodrop (results below). Froze RNA sample.
Concentration 193.9 ng/uL
260 4.849
280 2.681
260/280 1.81
260/130 1.94
Next Steps: Reverse transcription and PCR based on primers picked previously. My primers are for VtpA metalloprotease to look at Vibrio pathogenicity.
October 6, 2009
Lab 1: Tissue Extraction I
Received 2 pre-weighed tissue samples (50-100 mg for RNA isolation and 25 mg for protein extraction). My tissue is Vibrio tubiashii that has been in the presence of oysters.
I. RNA Isolation
To tissue, added 500 uL of TriReagent. Homogenized tissue in TriReagent with disposable pestle until tissue was broken up. Added 500 uL more of TriReagent. Vortexed for 15s. Labeled with initials and date and gave to MG to store at -80.
II. Protein Extraction
Added 500 uL of CelLytic MT solution to tissue sample. Homogenized tissue in solution with disposable pestle. Inverted tube several times to mix. Put tube in refrigerated microcentrifuge to spin at max speed for 10 minutes. When done spinning, transferred supernatant to clean tube labeled "Protein" (+ date + initials) and put on ice. In a new tube, added 15 uL of DI water and 15 uL protein sample (1:2 dilution of protein). Added 1.5 mL of Bradford reagent to protein, inverted to mix and let incubate at room temperature for about 20 minutes. In parallel, made a control blank with 30 uL DI water and 1.5 mL Bradford reagent.
Mixed both tubes and transferred to disposable cuvettes with pipette. Set spectrophotemeter to 595 nm. Used control to set blank then measured absorbance of protein sample. Mixed both blank and protein with pipette and repeated. Both measured absorbances for the sample were 0.106.
Accounting for the 1:2 protein dilution, the total protein concentration is 1011.9(0.106)x2 = 214 ug/mL.
Gave protein sample to MG to store at -20.
Next steps: Based on tissue sample, will decide what physiology/process want to study in lab and which loci will be appropriate for this study.