Christin's+Notebook


 * 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:

EU638304 295 - 211 107 - 191 All Exon
 * || 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__

(Completed by Sam White)
 * Extraction of DNA from Gel**

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)



__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

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
 * __Master Mix__**


 * 1) genomic DNA Grey Whale 1 ul
 * 2) genomic DNA Fin Whale 1 ul
 * 3) Neg Control 1 ul

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
 * __Master Mix__**


 * 1) genomic DNA Grey Whale 1 ul
 * 2) genomic DNA Fin Whale 1 ul
 * 3) Neg Control 1 ul

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
 * __Master Mix__**


 * 1) genomicDNA Grey Whale 1 ul
 * 2) genomic DNA Fin Whale 1 ul
 * 3) Neg Control 1 ul

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
 * __Master Mix__**


 * 1) genomic DNA Grey Whale 1 ul
 * 2) genomic DNA Fin Whale 1 ul
 * 3) Neg Control 1 ul

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
 * Denaturization and Annealing**

__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



- **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 Gel with Grey and Fin Whale DNA** **with Ladder this time :0)**
 * Run PCR on Grey and Fin Whale DNA with designed primers (same as before: __20090115__)

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



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
 * Cut out Lanes:**


 * 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__

EG329058 - Ordered Cet_GStra_F and R EG329066 - Ordered Cet_HSP6_F and R DT661080 - Ordered Cet_ SD_F and R
 * Run PCR with designed primers (see below) on Grey whale and Fin whale DNA**

Cet_COX2_F Cet_COX2_R
 * Quatitation of leukocyte gene expression in cetaceans (Sitt et al. 2008)**

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
 * __Master Mix__**


 * 1) genomic DNA Grey Whale 1 ul
 * 2) genomic DNA Fin Whale 1 ul
 * 3) Neg Control 1 ul

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
 * __Master Mix__**


 * 1) genomic DNA Grey Whale 1 ul
 * 2) genomic DNA Fin Whale 1 ul
 * 3) Neg Control 1 ul

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
 * __Master Mix__**


 * 1) genomicDNA Grey Whale 1 ul
 * 2) genomic DNA Fin Whale 1 ul
 * 3) Neg Control 1 ul

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
 * __Master Mix__**


 * 1) genomic DNA Grey Whale 1 ul
 * 2) genomic DNA Fin Whale 1 ul
 * 3) Neg Control 1 ul

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
 * Denaturization and Annealing**

__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)

__20090112__

DNA purification of Fin whale tissue using DNeasy tissue kit Start incubation at 55 degrees C at 1410

__20090108__

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
 * Design Fwd and Rv Primers for:**

__20081205__

Begin the rest of the steps for Purification of DNA from Gray Whale

Run a Spec. on 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