Kulper,+Tyler

Zebrafish Genetics

9/1/11

Lab 1: Overview of transgenesis strategy, genomic DNA isolation, and bioinformatics [|Lab 1 DNA isolation & bioinformatics.pdf]

1.) Anesthetized zebrafish in 200 mg/L tricaine 2.) Removed head and tail, chopped remaining body into pieces 3.) Snap froze tissue with liquid nitrogen, made into a powder with mortar and pestle 4.) Added powdered tissue to 7.5 ml of lysis buffer 5.) Incubated on rocking platform for 45 min at room temperature 6.) Transferred solution into 18 ml of EtOH, positioning it below the EtOH with a pipette 7.) Used Shepherd's crook to extract DNA from interface of solution 8.) Incubated DNA in 1 ml of TE in cold room

CNS for T-box transcription factor 5:

>danRer4_dna range=chr5:75673910-75674273 5'pad=150 3'pad=150 strand=+ repeatMasking=none CTGCTCATCTCAGGGAAAACACACACACACACACATGCACACGCACACAC ACACACACGCACGCACGCCATATTTACACCTCCACCCCTGCTTTCGGCGT TCACTCCTGCATTTCAGCAGTCTTGCCTTTCATGTGTTTCAGTCTGAGCA CACACACACACACACACACACACACACACACACACACACACACACACACA CACACACACACACACACACACACACAGATTTGGCAGACGATCAAAAGAAC AGTCAAATCTGCTGTAACACAAAGCTGCTGTGAACTGTTGGCTGATGTGT GTTACAACCTGTTGATCGAGCTCAGTTTGGAGAAGCAGAGACTCCTCAGC ACAGAAACACCGTC

9/6/11

Lab 2: Genomic DNA characterization [|Lab 2 DNA characterization.pdf]

1.) Prepared 1.0% agarose gel by heating 0.5g agarose in 50 mL TAE buffer in microwave. 2.) Added GelGreen DNA stain (2.5 microliters) 3.) Poured dissolved agarose into mold with comb. Solidified after ~15 min. 4.) Placed gel in electrophoresis rig and covered with TAE buffer 5.) Loaded 5 microliters of DNA ladder into second well 6.) 10 ul of our zebrafish DNA was combined with 2 ul of 6x TriTrack loading dye. Mixture was loaded into third well. 10 ul of the control DNA was also combined with 2 ul of the loading dye, and was loaded into the fourth well. 7.) Electrophoresed gel at 100v

Our agarose concentration was too high. We used 1.0% instead of 0.5%. Our DNA did not show up in the gel when it was electrophoresed. A picture was not taken of the gel.

9/8/11

Changed gene to **PITX2**. Dr. Balza did not think the CNS that was found for T-box Transcription factor 5 was satisfactory. PITX2 was found to have multiple CNS's.

PITX2: []

CNS 1: []

CAAGATTCCGGGTGACTGATAAAAAGTGACTTAATTAGGGAACCTAACTG AGTTTGACACCACCACCCATATCATTTATCTAGCCTACCTAAAACAAACG GAGCAGTCCGTGTTGTTCTAACTCGATGACAGTCTTGCATTGCTTTAGAT AGACTAATTAATTATGTAGGCTAATTCTTTTCTACAATTGTTGCAGGTGT [ GGTTCAAGAATCGACGGGCAAAATGGAGAAAAAGGGAGAGGAATCAACAG GCTGAGCTGTGTAAAAACGGTTTCGGCCCTCAATTCAACGGACTTATGCA GC ]CCTACGATGATTGGGGGGGGGGGGGTAATGAGACAGGAAGCGAAGTGG GAGAGAGAGAGAGGGGGTAGGGTAGGGAAATGCCCTCGACCCGTGATTCG AACCCGCAACGCCCTGACGTGCTATTGCACCATATGTCGGCGTGCTAACC ACTAGGTTATTGCGGCGACTGAAATGTGCATTTTTTAATCAATTTTTGGC GT

Left Primer: GGAGCAGTCCGTGTTGTTCT Tm: 60.31 degrees Celsius

Right Primer: CGCTTCCTGTCTCATTACCC Tm: 59.69 degrees Celsius

Sequence Size: 502

Primer Info: [|PITX2 CNS1 Primers.docx]

CNS 6: []

TCTCTTTGCTTAGCCAAGCATTTTAAGATCCATAATTTGTGTACACACTC CCTAATCCCCTGTATTTAAGTCCATGAGCGGCCATTTCTGCTATCTCCAT CTCTCTTTTCTGTTTGTTAGCGATCGCCCATAATACACAGCGCACCCACA GCGCATGATAAGCACATTTATATCGCCCTTCCTTCGCCTGGTGTTGGACT [ AGAGCTCGTCTAAATCCGGTGGAAGCTTAATGTTTATCCGACGTGAAAAT AATTACTCAAAAGTGTTTCTCTTAATCCGAATAATGAGGGGCCCAGAGTA GGGCTCAGTGTCATTGTAAATCAGGACAGGGCGATTGACTCCTCGCTCTT GTAAGTTTCAGGTGAGGATGGGGCCGGATAAAGGTTAGCTTAGGCCTAAT CCAGCCACGCTGCAGCCCTTAATCTGTTATGATACAAGGATGATCACTTA  AAA ]AGGGAGAAGGCCGTTTGACTGGGACCAGTCTTAAAGCATGCTTAGTG GCCCGCTGTCTCCGCAGTTGCAGAAAAAGCTCTTTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTAGGGCTGGAAGAAATGAATGAAAACATCT CAATATCAACTCACTGCACGCTAATTGTTTGAGGGGCTGTGATTGAGGTG GAG

Left Primer: TTCGCCTGGTGTTGGACT Tm: 60.25 degrees Celsius

Right Primer: CAGTCAAACGGCCTTCTCC Tm: 60.79 degrees Celsius

Sequence Size: 653

Primer Info: [|PITX2 CNS6 Primers.docx]

9/15/11

1.) Spun primers in centrifuge at 6500 RPM for 1 minute 2.) Diluted primers in ~300 uL molecular biology grade water (nmol x 10) to make 100 uM stock solution. Each was then vortexed 3.) Made a 10 uM primer working solution by adding 10 uL of left primer and 10 uL of right primer to 80 uL of molecular biology grade water. Solution was vortexed 4.) Made two master mixes for each of my CNS's by mixing 5 times each amount -105 uL molecular biology grade water -12.5 uL 10x Accuprime polymerase buffer II -2.5 uL Genomic DNA in TE -2.5 uL Primer mix working sol -2.5 uL Accuprime Taq polymerase 5.) Separated the mixes into four PCR tubes each, each tube having 25uL 6.) CNS 1 was labeled as 5, CNS 6 was labeled as 7 (based on the last digit from the Ref. No. on my left primers found on the IDT Oligonucleotide Specification Sheet) 7.) Each set of tubes was labeled 1 through 4, and placed into the thermocycler for PCR. In the thermocycler: 1 = 60 degrees Celsius 2 = 59.8 degrees Celsius 3 = 59.2 degrees Celsius 4 = 58.3 degrees Celsius 8.) Thermocycler ran for ~2 hours. Reactions were placed in freezer at -20 degrees Celsius once finished