Capstone Time line:
- Samples of Grey whale tissue obtained from Jina Ylitalo, NOAA (October 30, 2008)
- Samples of Fin whale tissue obtained from Greg Jensen (October 30, 2008)
- Extraction of DNA from grey and fin whale tissue (December 10, 2008)
- Identify immune related genes in cetaceans; create database
- published genes (corenucleotide identified via literature) (December 15, 2008)
- ESTs identified via bioinformatic algorithims (December 30, 2008)
- Design primers based on genes (5-10) identified above and amplify homologs in Grey and Fin whale DNA samples (January 30, 2009)
- DNA sequence products (February 15, 2009)
- Characterize sequences (ie alignments, genomic structure (March 15, 2009)
- Develop quantitative PCR assay for 1-3 genes (March 30, 2009)
- Complete Draft of Presentation (April 30, 2009)
- Complete Draft of Scientific Paper (May 15, 2009)
20090305
Annotated sequences with top BLAST hits on NCBI:
| Accession Number |
HIT |
QUERY |
Introns/CDS Position |
|
| F Whale_Gstra_R |
AY878121 |
13,195 - 13,397 |
321 - 526 |
|
| AL121969 |
102,140 - 101,938 |
321 - 526 |
102,141 - 101,938 |
|
| AF050057 |
193 - 267 |
1 - 76 |
220 - 268 193 - 220 |
|
| G Whale_Gstra_F |
AL121969 |
100,744 - 101,127 |
1 - 329 |
100,757 - 101,128 100,743 - 100,757 |
| AY878121 |
14,591 - 14,026 |
1 - 512 |
14,025 - 14,578 14,590 - 14,578 |
|
| G Whale_SDo_R |
BC122671 |
1,157 - 1,060 |
111 - 208 |
All Intron |
| DQ146944 |
1,063 - 1,968 |
13 - 208 |
All Intron |
|
| G Whale_COX2_F |
AY229989 |
4,047 - 4,348 |
2 - 307 |
4,047 - 4,049 |
| AF027335 |
3,503 - 3,778 |
5 - 308 |
3,502 - 3,513 |
|
| G Whale_COX2_R |
AF031699 |
5,679 - 5,434 |
16 - 263 |
5,505 - 5,433 5,505 - 5,590 5,590 - 5,680 |
| AF027335 |
4,948 - 4,703 |
16 - 259 |
4,702 - 4,773 4,773 - 4,859 |
|
| AY382629 |
5,623 - 5,431 |
71 - 261 |
5,431 - 5,504 5,504 - 5,587 |
|
| DQ173702 |
453 - 384 |
105 - 174 |
452 - 453 383 - 452 |
|
| EU638321 |
295 - 211 |
107 - 191 |
All Exon |
|
| EU638303 |
295 - 211 |
107 - 191 |
All Exon |
20090224
Extraction of DNA from Gel
(Completed by Sam White)
Procedure to use Ultrafree-DA
1. Place gel slice into Gel Nebulizer and seal device with the cap attached to vial
2. Spin at 5,000 x g for 10 minutes, Centrifugation forces the agarose through the gel nebulizer, converting it to a fine slurry that is captured by Ultrafree-MC. Extruded DNA in elerctrophoresis buffer passes through the microporous membrane in Ultrafree-MC and collects in the filtrate vial.
3. Discard the Ultrafree-MC and gel nebulizer. You can seguence or clone the DNA in the filtrate without further purification.
Obtained sequences and placed into a file(F and G Whale) in Geneious.
Alignment was completed on Cet_IL17_F &R and was Blast through NCBI and found a match with a Human IL17.
Alignments on the other sequences where done as well.
20090203
Run Gel with designed primers on Grey and Fin Whale DNA
1.5 g Agarose/ 100 mL TAE
6 ul Ethidium Bromide
Run for 1hr at 115 volts
Medium gel plate with 20 well 1.0mm comb
25 ul product
25 ul 100 bp ladder
14 lanes:
1. 100 bp Ladder
2. Negative Control Cet_IL17_ F/R
3. Grey DNA Cet_IL17_F/R
4. Fin DNA Cet_IL17_F/R
5. Negative Control Cet_IL13_F/R
6. Grey DNA Cet_IL13_F/R
7. Fin DNA Cet_L13_F/R
8. Negative Control Cet_IL2_F/R
9. Grey DNA Cet_IL2_F/R
10. Fin DNA Cet_IL2_F/R
11. Negative Control Cet_TLR5_F/R
12. Grey DNA Cet_TLR5_F/R
13. Fin DNA Cet_TLR5_F/R
14. 100bp ladder
Picture of gel
5 separate bands were cut out and place in labeled microcentrifuge tube.
In lane:
3. Grey DNA Cet_IL17_F/R
4. Fin DNA Cet_IL17_F/R
6. Grey DNA Cet_IL13_F/R
7. Fin DNA Cet_IL13_F/R (2 separate bands were cut out)
 |
| external image |
20090202
Run PCR on Grey and Fin Whale DNA with designed primers
1. Cet_IL17_F & R; Base Pairs (bp) 324
2. Cet_IL13_F & R; bp 279
3. Cet_IL2_F & R; bp 287
4. Cet_TLR5_F & R; bp 365
Master Mix
2x Go TAQ Mm 12.5 ul x 4 = 50.0 ul
Cet_IL17_F 0.5 ul x 4 = 2.0 ul
Cet_IL17_R 0.5 ul x 4 = 2.0 ul
H2O 10.5 ul x 4 = 42.0 ul
- genomic DNA Grey Whale 1 ul
- genomic DNA Fin Whale 1 ul
- Neg Control 1 ul
Master Mix
2x Go TAQ Mm 12.5 ul x 4 = 50.0 ul
Cet_IL13_F 0.5 ul x 4 = 2.0 ul
Cet_IL13_R 0.5 ul x 4 = 2.0 ul
H2O 10.5 ul x 4 = 42.0 ul
- genomic DNA Grey Whale 1 ul
- genomic DNA Fin Whale 1 ul
- Neg Control 1 ul
Master Mix
2x Go TAQ Mm 12.5 ul x 4 = 50.0 ul
Cet_ IL2_F 0.5 ul x 4 = 2.0 ul
Cet_ IL2_R 0.5 ul x 4 = 2.0 ul
H2O 10.5 ul x 4 = 42.0 ul
- genomicDNA Grey Whale 1 ul
- genomic DNA Fin Whale 1 ul
- Neg Control 1 ul
Master Mix
2x Go TAQ Mm 12.5 ul x 4 = 50.0 ul
Cet_ TLR5_F 0.5 ul x 4 = 2.0 ul
Cet_ TLR5_R 0.5 ul x 4 = 2.0 ul
H2O 10.5 ul x 4 = 42.0 ul
- genomic DNA Grey Whale 1 ul
- genomic DNA Fin Whale 1 ul
- Neg Control 1 ul
Denaturization and Annealing
95 degrees C - 10 minutes
95 degrees C - 45 sec x 40
55 degrees C - 1 minute x 40
72 degrees C - 1 minute x 40
72 degrees C - 10 minutes
20090120
Run gel with genomic DNA and negative controls for each set of primers
0.5 Agarose/ 50mL !% TAE
1 ul ethdium bromide/ 10mL = 5 ul of E.B,
1 Lanes:
1. Neg control Cet_GStra_F and R
2. Grey Whale Cet_GStra_F and R
3. Fin Whale Cet_GStra_F and R
4. Neg control Cet_HSP6_F and R
5. Grey Whale Cet_HSP6_F and R
6. Fin Whale Cet_HSP6_F and R
7. Neg control Cet_ SD_F and R
8. Grey Whale Cet_ SD_F and R
9. Fin Whale Cet_ SD_F and R
10. Neg control Cet_COX2_F and R
11. Grey Whale Cet_COX2_F and R
12. Fin Whale Cet_COX2_F and R
 |
| Gel of G/F whale DNA |
- Section of DNA was cut out of the gel
- 1st two bands: Grey DNA Cet_GStra_F/R, Fin DNA Cet_GStra_F/R
- Last two bands: Grey DNA Cet_COX2_F/R, Fin DNA Cet_COX2_F/R
Run PCR on Grey and Fin Whale DNA with designed primers (same as before: 20090115)
Run Gel with Grey and Fin Whale DNA with Ladder this time :0)
1st Lane:
1. 100 bp ladder
2. Neg Control Cet_GStra_F and R
3. Grey Whale Cet_GStra_F and R
4. Fin Whale Cet_GStra_F and R 4.
5. Neg control Cet_HSP6_F and R
6. Grey Whale Cet_HSP6_F and R
7. Fin Whale Cet_HSP6_F and R
8. Neg control Cet_ SD_F and R
9. Grey Whale Cet_ SD_F and R
10. Fin Whale Cet_ SD_F and R
2nd Lane:
1. 100 bp ladder
2. Neg control Cet_COX2_F and R
3. Grey Whale Cet_COX2_F and R
4, Fin Whale Cet_COX2_F and R
 |
| external image |
Cut out Lanes:
3. Grey Whale Cet_GStra_F and R
4. Fin Whale Cet_GStra_F and R
6. Grey Whale Cet_HSP6_F and R
9. Grey Whale Cet_ SD_F and R (1st and 2nd band)
10. Fin Whale Cet_ SD_F and R
3. Grey Whale Cet_COX2_F and R
4, Fin Whale Cet_COX2_F and R
Designed and ordered primers
DV799566 - Ordered Cet_IL17_F and R
DV799568 - Ordered Cet_IL13_F and R
DV799577 - Ordered Cet_IL2_F and R
DV799591 - Ordered Cet_TLR5_F and R
20090115
Run PCR with designed primers (see below) on Grey whale and Fin whale DNA
EG329058 - Ordered Cet_GStra_F and R
EG329066 - Ordered Cet_HSP6_F and R
DT661080 - Ordered Cet_ SD_F and R
Quatitation of leukocyte gene expression in cetaceans (Sitt et al. 2008)
Cet_COX2_F
Cet_COX2_R
Master Mix
2x Go TAQ Mm 12.5 ul x 4 = 50.0 ul
Cet_GStra_F 0.5 ul x 4 = 2.0 ul
Cet_GStra_R 0.5 ul x 4 = 2.0 ul
H2O 10.5 ul x 4 = 42.0 ul
- genomic DNA Grey Whale 1 ul
- genomic DNA Fin Whale 1 ul
- Neg Control 1 ul
Master Mix
2x Go TAQ Mm 12.5 ul x 4 = 50.0 ul
Cet_HSP6_F 0.5 ul x 4 = 2.0 ul
Cet_HSP6_R 0.5 ul x 4 = 2.0 ul
H2O 10.5 ul x 4 = 42.0 ul
- genomic DNA Grey Whale 1 ul
- genomic DNA Fin Whale 1 ul
- Neg Control 1 ul
Master Mix
2x Go TAQ Mm 12.5 ul x 4 = 50.0 ul
Cet_ SD_F 0.5 ul x 4 = 2.0 ul
Cet_ SD_R 0.5 ul x 4 = 2.0 ul
H2O 10.5 ul x 4 = 42.0 ul
- genomicDNA Grey Whale 1 ul
- genomic DNA Fin Whale 1 ul
- Neg Control 1 ul
Master Mix
2x Go TAQ Mm 12.5 ul x 4 = 50.0 ul
Cet_ COX2_F 0.5 ul x 4 = 2.0 ul
Cet_ COX2_R 0.5 ul x 4 = 2.0 ul
H2O 10.5 ul x 4 = 42.0 ul
- genomic DNA Grey Whale 1 ul
- genomic DNA Fin Whale 1 ul
- Neg Control 1 ul
Denaturization and Annealing
95 degrees C - 10 minutes
95 degrees C - 45 sec x 40
55 degrees C - 1 minute x 40
72 degrees C - 1 minute x 40
72 degrees C - 10 minutes
20090113
Let Fin whale sample sit over night in incubation temp of 55 degree C
Took sample out of incubation at 1120
Completed DNaesy tissue kit
Run a spec. for Fin whale DNA
49.51 ng/uL - DNA50 (other was run incorrectly using RNA spec)
 |
| Fin whale DNA spec 49.51 ng/uL |
20090112
DNA purification of Fin whale tissue using DNeasy tissue kit
Start incubation at 55 degrees C at 1410
20090108
Design Fwd and Rv Primers for:
EG329058 - Ordered Cet_GStra_F and R
EG329066 - Ordered Cet_HSP6_F and R
DT661080 - Ordered Cet_ SD_F and R
DV799566
DV799568
DV799591
Cox2 Fwd and Rv Primers from (Sitt et al. 2008) - Ordered Cet_cox2F and R
20081205
Begin the rest of the steps for Purification of DNA from Gray Whale
Run a Spec. on Oyster and gray whale DNA
 |
| Spec. of Oyster and gray whale DNA |
20081204
Began Purification of DNA from Gray Whale tissue with DNeasy Tissue Kit
Let sample incubate overnight at 55 degrees Celsius
20081204
Gel run on Oyster Tissue
0.5 Agarose/50 mL 1% TBE
1 ul E.B./10mL of solution
Tray:
- 100 bp ladder
- Negative Control w/IL17 150-0 51 & 31
- IL17 150-0 51 &31
- Negative Control w/IL17 Fw & Rv
- IL17 Fw and IL17 Rv
Picture of Gel Oyster tissue
20081125
DNeasy Tissue Kit
Purification of DNA from Oyster tissue
PCR of Oyster Tissue
20081118
Website that contains Cetacean EST's
http://tinyurl.com/6r4u97