Schulz,+Rebecca

Lab 1: Overview of Transgenesis Strategy, Genomic DNA Isolation, and Bioinformatics Partners: Rachel Nolte and Tyler Kuiper Assigned Gene: SRF
 * September 1st, 2011**

Lab Goals: 1) Learn about lab techniques we'll be using this semester 2) Extract isolated DNA from a Zebrafish 3) Use Vista website for comparative genomics to find putative enhancer sequence of assigned gene 4) Design primer sequence 5) Become familiar with online research tools concerning genes and genetic diseases

Safety: Wear lab goggles and gloves when handling liquid nitrogen. Gloves are required when handling anything concerning DNA. Refer to Lab 1 handout for more specific/further safety instructions. Materials: Refer to Lab 1 handout (page 4) for materials used in this lab.

Procedure: 1. Anesthetize one zebrafish in tricaine until completely sedated. //The whole class shared beakers of anesthetization and we shared one fish per group of 3//.

2. Kill the fish via decapitation. Also a good idea to cut off fins. Cut the fish into small pieces. //Tyler said it was harder to decapitate and cut the fins because of the excess liquid we had in our petri dish.//

3. Freeze the tissue in liquid nitrogen and pulverize to a powdery substance using a pre-frozen mortar and pestle. //Dr. Balza pre-froze the mortars and pestles for us in the -80 degree freezer. It was difficult to crush the fish pieces up, but we did finally gain a powder.//

4. Once the liquid nitrogen has evaporated, add the powdered tissue to 7.5 mL of lysis buffer in a 50mL conical tube. Let the powder sit on the buffer surface, then shake to submerge the powder.

5. Incubate mixture for 45-60 minutes at room temperature on a rocking platform.

6. Transfer the lysis buffer solution into a new 50mL conical tube which already has 18mL of 190 proof ethanol (EtOH) in it. CAREFULLY layer the lysis buffer solution UNDER the EtOH. //There was some confusion about this, but the lysis solution must be under the ethanol to make an interface for the DNA.//

7. Make a Shepherd's crook by warming the tip of a glass pipette in a Bunsen burner and bending to form a hook. Use the Shepherd's crook to slowly stir the interface and collect the DNA in a gelatinous mass.

8. Remove the crook, with its attached DNA, and allow some EtOH to drain away. The DNA should be tightly packed. Wash the DNA off the crook with 5mL EtOH into a fresh tube. //We actually did not wash the DNA with more ethanol. We had the DNA on the crook and went directly to the next step...//

9. Transfer to a fresh tube containing 1mL of TE. Allow the DNA to rehydrate overnight at 4 degrees Celsius.

~VISTA Browser for Comparative Genomics~ Could not locate SRF gene in zebrafish genome. Dr. Balza suggested Nkx2.5 (his second-favorite gene). [|NKX2.5.pdf] CNS follows the gene itself.

Lab 2: Genomic DNA Characterization Partners: Rachel Nolte and Tyler Kuiper
 * September 6th, 2011**

Lab Goal: Discover the quality and quantity of DNA isolated from zebrafish in Lab 1.

Safety: See page 1 in Lab 2 Handout. Materials: See page 1 in Lab 2 Handout.

Notes: [|Zebrafish_Lab_2_Notes_001.jpg]

Procedure: 1. Prepare an agarose gel (use chart on page 2 of Lab 2 Handout for % agarose suggestions). //We decided to make a 1% agarose gel...so we dissolved 0.5g of agarose in 50mL of 1xTAE using the microwave.//

2. Add GelStar or GelGreen DNA stain to agarose mixture: 2.5 microliters per 50mL gel.

3. Pour the dissolved agarose into the mold and put the comb in one end to create wells. Make sure there are no air bubbles. Wait for the gel to harden...a cloudy appearance is a good indicator.

4. Once hardened, take the comb out and submerge the gel in 1xTAE buffer in an electrophoresis rig. To conserve buffer use enough to cover the gel.

5. Load 5 microliters of the DNA ladder into the first lane of the gel. //We loaded ours into the second well, just to centralize our samples and in case of accidental tears to the gel. Also, we used 12 microliters of DNA ladder since our other wells would be filled with as much.//

6. Combine 5 microliters of your DNA solution from Lab 1 with 1 microliter of 6x TriTrack loading dye. Do the same to the control DNA. //To maximize our results, we used 10 microliters of each DNA sample. Each sample of DNA was then combined with 2 microliters total of loading dye. Thus, 12 microliters per well. We put our DNA in the 3rd well, and the control DNA in the 4th well. We ended up repeating this step (read further please), and loaded a second sample of our DNA in the 5th well, and more control DNA in the 6th well.//

7. Make sure the wells containing DNA are near the black electrode (which has a negative charge and will repel DNA, also negatively charged because of its phosphate backbone), and the base of the gel is toward the red electrode (which has a positive charge and so will attract the negatively charged DNA). Run the gel at ~100v for about an hour, or until the bromophenol blue is near the end of the gel. //Here's where we had some issues. Everyone else in the class seems to have used the 0.5% agarose gel, whereas we used 1%. A higher concentration allowed us to observe a bolder line in the DNA ladder lane, but no DNA after an hour in the other two lanes. This is where we repeated step 6, reloaded the same gel, and underwent electrophoresis once more, letting the gel run for 2 hours. Once the two hours were up, we looked at our gel once more. The ladder had traveled only slightly further, and control DNA was now thinly visible in Lane 4, slightly drawn out, which indicated it had degraded with further electrophoresis. In our Lane 6, with the second batch of control DNA, the well was dark with DNA present, but it had hardly penetrated into the gel, if at all. We did not see any of our DNA during imaging. Part of this may be due to loading the second batch in. When I was loading the well, I stuck the pipette tip too far into the well and the sample pooled deeper in the gel and so would not electrophorese properly. As for our Lane 2 DNA, there had been no problems in loading it, but none showed up during the imaging process. The reason our DNA did not run far into the gel (control DNA, that is), is because we used a higher concentration of agarose. Genomic DNA, as we learned in the lecture session, is in a large chunk and so should be a dark, thick band near the wells of the gel (see link to my notes). So we don't actually know for certain whether or not we have any DNA from last week's lab...//

Finding the DNA Primer for Nkx2.5 on Zebrafish
 * September 8th, 2011**

CNS for Nkx2.5: chr14:27,406,115-27,406,621 (507bp) [|Nkx2.5 CNS.pdf]

CNS DNA (+approx. 200bp surrounding CNS): >danRer4_dna range=chr14:27406115-27406621 5'pad=0 3'pad=0 strand=+ repeatMasking=none CATAATCCATACCCATCCATATTGATATGTTGATCGGAGTTTCACAAAAA AAGTGAAAAGATTATACAGTCTGTATGTGCATCTCTCTTCCTCTTTTTAA GAACACTTATCTTTCAGTCATTTCCTATAAGCCTATATTTTGGTTAATTT ACTCAAAAGAAAGGAAAAAGTCTTTTCTTCAGAATATAGAGGACTGTAGC TAGGAAGAGAGCAAGAGCGAGGAAAATAAGCAGCTTATCTGTAAGTTCCC GTCGGTTGTATTTAATGATGAGCTTTCTGCCCAGCTGTATAGTTCCTGTC ATGGCTTTAAACTCTTCATGAGTTTCCAGAACAGTCCTGGAAGATGTTGC TAAAGGTGTGGAACAAACAGGAACACATTAGTTTCTCAGCATTCAGACAG GGCTTAAATAGAAAAAGAGGCTACTGTGGGATGATCAAAAACTAAAAACA TCATTCAGCAAATAAACGTAACTTGGAATGCACTGGTATGGATATTTGGG TTGATAC

JUST CNS: chr14:27,406,286-27,406,449 (164bp) [|JUST CNS DNA.pdf]

JUST CNS DNA: >danRer4_dna range=chr14:27406286-27406449 5'pad=0 3'pad=0 strand=+ repeatMasking=none CTTTTCTTCAGAATATAGAGGACTGTAGCTAGGAAGAGAGCAAGAGCGAG GAAAATAAGCAGCTTATCTGTAAGTTCCCGTCGGTTGTATTTAATGATGA GCTTTCTGCCCAGCTGTATAGTTCCTGTCATGGCTTTAAACTCTTCATGA GTTTCCAGAACAGT

Whole Thing: >danRer4_dna range=chr14:27406115-27406621 5'pad=0 3'pad=0 strand=+ repeatMasking=none CATAATCCATACCCATCCATATTGATATGTTGATCGGAGTTTCACAAAAA AAGTGAAAAGATTATACAGTCTGTATGTGCATCTCTCTTCCTCTTTTTAA GAACACTTATCTTTCAGTCATTTCCTATAAGCCTATATTTTGGTTAATTT ACTCAAAAGAAAGGAAAAAGT [CTTTTCTTCAGAATATAGAGGACTGTAGC TAGGAAGAGAGCAAGAGCGAGGAAAATAAGCAGCTTATCTGTAAGTTCCC GTCGGTTGTATTTAATGATGAGCTTTCTGCCCAGCTGTATAGTTCCTGTC ATGGCTTTAAACTCTTCATGAGTTTCCAGAACAGT] CCTGGAAGATGTTGC TAAAGGTGTGGAACAAACAGGAACACATTAGTTTCTCAGCATTCAGACAG GGCTTAAATAGAAAAAGAGGCTACTGTGGGATGATCAAAAACTAAAAACA TCATTCAGCAAATAAACGTAACTTGGAATGCACTGGTATGGATATTTGGG TTGATAC

PRIMERS: Forward (Left) Primer: TGGTTAATTTACTCAAAAGAAAGGAAA Reverse (Right) Primer: TGTGTTCCTGTTTGTTCCACA Left Primer T m : 59.86 degrees Celsius Right Primer T m : 60.03 degrees Celsius SEQUENCE SIZE: 507bp [|Primer3 Output.pdf]

__in silico PCR (UCSC) __ >[|chr14:27406255+27406491] 237bp TGGTTAATTTACTCAAAAGAAAGGAAA TGTGTTCCTGTTTGTTCCACA TGGTTAATTTACTCAAAAGAAAGGAAAaagtcttttcttcagaatataga ggactgtagctaggaagagagcaagagcgaggaaaataagcagcttatct gtaagttcccgtcggttgtatttaatgatgagctttctgcccagctgtat agttcctgtcatggctttaaactcttcatgagtttccagaacagtcctgg aagatgttgctaaaggTGTGGAACAAACAGGAACACA

__Primer Melting Temperatures __ 



The temperature calculations are done assuming 50 mM salt and 50 nM annealing oligo concentration. The code to calculate the melting temp comes from Primer3.