Pesch,+Kayli

__**Zebrafish Genetics- GATA-4**__

9/1/11 I did not participate in lab because of an interfering class and the want to preserve as much sacrifice of fish as possible. My classmates completed lab with the following procedure (copied from Emily Bolda) and harvested the DNA with which I will continue lab procedures.

1. anesthetized a zebrafish with 200mg/l then with Angie and Selciya we chopped off the head. 2.removed the tail, some fins, and took off a few scales. We chopped the fish into pieces and froze with some liquid nitrogen. 3. crushed up our fish pieces with a chilled mortar and pestle. Our fish did not turn into powder, more mush so we froze a second time. It still was mushy so we poured our 7.5ml of lysis buffer into the mortar and mixed around with our spatula. This worked to get most of the fish remains out and into our conical tube. 4.placed it on a rocking platform to incubate for 45-60mins at room temp. 5. transferred the lysis buffer solution into a conical tube(18ml of 190 proof EtOH) but our buffer got very mixed up when placing it into the tube with EtOH so there was not a clear layer of separation to see the DNA.could cause chances of not working. 6. used another lab groups DNA, catching with a "shepard's crook" (not sure if much was left) 7. took out crook and waited a few seconds for EtOH to evaporate.should have touched to Kimwipe to help before rinsing in 5ml EtOH 8. transferred into pipette with 1ml TE.placed in box in fridge. 9. went to compare zebrafish and human sequence homology for my gene (MEF2c) and looked at sequence. unsure where to zoom and label the CNS regions.will have to have ask for help to go farther.

9/6/11

Lab2: Genomic DNA Charactaracterization 1. Mix approximately 0.25g agarose in 50mL 1xTAE 2. Microwave mixture 45sec until agarose is dissolved 3. Add 2.5uL GelGreen DNA stain 4. Pour dissolved agarose into gel mold and place comb in one end. Wait until solid (10min) 5. At this point, I noticed large dust particles in the gel that would inhibit the DNA from flowing through it so I tossed the gel into the garbage and repeated steps 1-4 6. After waiting for the gel to solidify for over 30min, the gel felt solid to the touch and had a cloudy appearance. 7. I carefully pulled the comb out of the gel and realized the gel had not solidified completely and the wells collapsed. 8. I looked on with Emily Bolda's group while they ran the electrophoresis and observed the results with them.

9/8/11

Identifying PCR Primers

1. Using VISTA, I isolated a CNS within a reasonable distance of the gata4 gene. 2. Then I used Primer 3 to identify reasonable primers. the process is outlined in the document below:



9/15/11

1. Received primers from Integrated DNA Technologies 2. Spin the microfuge tube with primer for 30sec 3. Dilute primers and briefly vortex -- a. Pesch GATA-R 24.5nm + 245uL nuclease free water -- b. Pesch GATA-L 28.8nm +288uL nuclease free water 4. Create Primer Working solution and vortex briefly. Store in -20C freezer -- a. Mix 10uL forward primer and 10uL antisense primer and 80uL nuclease free water

PCR 1. Prepare four 25uL PCR reactions in separate thin-walled PCR tubes with the following reagents -- a. 21.0uL Nuclease free water -- b. 2.5uL 10x Accuprime polymerase buffer II -- c. 0.5uL Genomic DNA in TE (~0.10ug) -- d. Primer mix working solution (previously prepared) -- e. Accuprime //Taq// polymerase 2. Insert PCR tubes into PCR machine and set for cycle 3. Store at -20C until further use
 * Temperature (In C) || Time (Min:Sec) || Cycles ||
 * 95 || :30 || 1 ||
 * 95 || :30 || 40 ||
 * 59.8, 59.2, 58.3, 57.0 || 1:30 || ' ||
 * 72 || :30 || ' ||
 * 72 || 2:00 || 1 ||
 * 4 || Indefinitely ||  ||

9/20/11 Agarose gel purification of PCR amplicons

1. Prepare agarose gel concentration- Heat and swirl until agarose is disolved -- a. 2.0% agarose = 50mL TAE buffer + 1mg agarose 2. Add 2.5uL GelStar to agarose solution 3. Pour gel and place comb in one end. Allow to solidify (~10min) 4. Remove comb and submerge in 1xTAE buffer 5. Load 5uL GeneRuler DNA 1Kb ladder to first lane of gel 6. Combine 5uL DNA solution (PCR product) with 1uL 6x TriTrack loading dye and load into lanes 2,3,4 and 5 7. Elextrophorese the gel at 100v until bromophenol blue is near the end of the gel (1hour 20min) 8. Image gel using Kodak IS4000MM image station. Add Photo of Gel 9. Excise the gel slice containing the amplicon band with a clean scalpel using UV light to highlight the amplicon 10. Put each slice into a microfuge tube and weigh to approximate the volume (1g = ~1mL) (Recorded in table below)** 11. Store at -20C until further use.


 * Label || Mass of Tube (g) || Mass of tube + sample (g) || Sample Mass (g) || x3 ||
 * 1 || 1.03 || 1.18 || 0.15 || 0.45 ||
 * 2 || 0.99 || 1.22 || 0.23 || 0.69 ||
 * 3 || 0.99 || 1.42 || 0.43 || 1.29 ||
 * 4 || 0.98 || 1.32 || 0.34 || 1.02 ||

The last row (x3) Is part of the next step.

9.22.11 Amplicon Extraction

1. Remove microtubes with gel cubes from freezer and bring to room temperature 2. Add 3x volume of sample of Buffer QX1 DNA binding solution to gel. Incubate 5min at 55C to dissolve agarose. (Recorded in Table above). 3. Resuspend the Qiaex II silica gel suspension by vortexing for 30sec. Add 10uL to each tube with gel slice. 4. Incubate at 50C for 10min to solubilize the agarose and bind the DNA to the silica. Mix by vortexing every 2min. 5. Centrifuge sample for 30sec and carefully digard the supernatent with a pipet 6.Wash the pellet with twice 500uL of wash buffer PE. Resuspend by vortexing, pellet with a brief centrifucation, and remove the supernatent with a pipet. 7.Wash the pellet with 500uL of wash buffer QX1. Resuspend by vortexing, pellet with a brief centrifugation, and remove the supernatent with a pipet. 8. Wash the pellet with twice 500uL of wash buffer PE. Resuspend by vortexing, pellet with a brief centrifucation, and remove the supernatant with a pipet.* 9. Air-dry the pellet for 10-15 min until pellet appears white. 10. Elute DNA into TE buffer. Re-suspend the pellet in a 30uL of sterile TE buffer and incubate the tube at 55C for 5min. Spin the tube and remove the supernatant into a new microfuge tube, taking care to avoid the pellet. 11. Store the DNA in a well-labeled tube at -20C


 * Steps 6-8 diverged from the outlined protocol slightly.

9.27.11 Cloning of the CNS amplicon (PCR product) into an entry vector using BP clonase

DNA extracted from lanes 1 and 2 were used for the following steps. 3 and 4 were continuously stored at -20C

1. Add the following to a 1.5mL microcentrifuge tube at room temperature: 7.5uL //att//B PCR product 0.5uL pDONR 221 (150 ng/uL) 2uL BP Clonase II enzyme/buffer mix 2. Vortex the 10uL briefly and incubate reaction at 25C for 1-2 hours. 3. Add 1uL of 2ug/uL Proteinase K solution and uncubate at 37C for 10min. 4. Store at -20C

9.30.11

1. Add 1uL of the BP clonase reaction to a microcentrifuge tube and place on ice to chill. 2. Thaw a vial of TOP10 E.coli cells on wet ice to chill. 3. When cells are thawed, mix cells by tapping gently. Add 20uL of cells to each chilled microcentrifuge tube containing DNA. One vial of cells is enough for two transformations. I shared one vial with Miranda W. and only used the DNA 1 (2 was stored at -20C) 4. Incubate the vial of cells + BP reaction product on ice for 30min. 5. Transform the DNA into the cells by heat-shocking the cells for 30sec at 42C without shaking. 6. Place the heat-shocked cells back into the ice for 2min. 7. Aseptically add 250uL of S.O.C. medium to the cells. 8. Shake at 225 rpm at 37C for 1 hour. 9. Spread 200uL of the transformed cells on a prewarmed kanamyacin plate. (to select for the successfully transformed colonies) 10. Incubate inverted plate overnight at 37C
 * Transformation of BP clonase reaction product into TOP10 chemically competent E. Coli**

10.1.11

Miranda came in to check our plates for bacteria colonies and she saw nothing but moved the plates into 4C cold room.

10.4.11

I checked my plate that was moved into 4C cold room and confirmed the lack of colonies. Plates were disposed of.

//I assumed something went wrong with the transformation step, so I continued another transformation into the E.coli with the entry vector prepared on 9.27.11//

10.7.11 Transformation of BP clonase reaction product into TOP10 chemically competent E. Coli

1. Add 1uL of the BP clonase reaction to a microcentrifuge tube and place on ice to chill. 2. Thaw a vial of TOP10 E.coli cells on wet ice to chill. 3. When cells are thawed, mix cells by tapping gently. Add 20uL of cells to each chilled microcentrifuge tube containing DNA. 4. Incubate the vial of cells + BP reaction product on ice for 30min. 5. Transform the DNA into the cells by heat-shocking the cells for 30sec at 42C without shaking. 6. Place the heat-shocked cells back into the ice for 2min. 7. Aseptically add 250uL of S.O.C. medium to the cells. 8. Shake at 225 rpm at 37C for 1 hour. 9. Spread 200uL of the transformed cells on a prewarmed kanamyacin plate. (to select for the successfully transformed colonies) 10. Incubate inverted plate overnight at 37C
 * One vial of cells is enough for two transformations. I used DNA 1 and 2 for two separate transformations and continued the steps with each separately.

10.8.11

I returned to check the plates for plates for colonies and saw none on either plate. I disposed of both plates.

//Continuing on with the product from PCR on 9.15.11 I attempted the transformation again. Running the PCR product through a gel once again showed the presence of DNA, but I took the extra step and quantified the DNA to make sure I had enough DNA for the transformation steps. This procedure is recorded below.// .

10.25.11

PCR 1. Prepare eight 25uL PCR reactions in separate thin-walled PCR tubes with the following reagents -- a. 21.0uL Nuclease free water -- b. 2.5uL 10x Accuprime polymerase buffer II -- c. 0.5uL Genomic DNA in TE (~0.10ug) (Stock) -- d. Primer mix working solution (previously prepared on 9.15.11) -- e. Accuprime //Taq// polymerase 2. Insert PCR tubes into PCR machine and set for cycle 3. Store at -20C until further use
 * Temperature (In C) || Time (Min:Sec) || Cycles ||
 * 95 || :30 || 1 ||
 * 95 || :30 || 40 ||
 * 55-60 || 2:00 || ' ||
 * 72 || :30 || ' ||
 * 72 || 2:00 || 1 ||
 * 4 || Indefinitely ||  ||

10.27.11 Agarose gel purification of PCR amplicons

1. Prepare agarose gel concentration- Heat and swirl until agarose is disolved -- a. 2.0% agarose = 50mL TAE buffer + 1mg agarose 2. Add 2.5uL GelStar to agarose solution 3. Pour gel and place comb in one end. Allow to solidify (~10min) 4. Remove comb and submerge in 1xTAE buffer 5. Load 5uL GeneRuler DNA 1Kb ladder to first lane of gel 6. Combine 5uL DNA solution (PCR product) with 1uL 6x TriTrack loading dye and load into lanes 2,3,4 and 5 7. Elextrophorese the gel at 100v until bromophenol blue is near the end of the gel (1hour 20min) 8. Image gel using Kodak IS4000MM image station. Add Photo of Gel 9. Only Lane 5 showed the presence of DNA.

10.29.11

Quantification of PCR product (from 9.15.11 and 10.27.11)

1. Prepare Quanti-IT BR working solution by diluting dsDNA BR reagent 1:200 into dsDNA buffer (Per sample) 2. Prepare sample for quantification by adding 2uL of purified pCR product to 198uL of Quant-IT BR working solution. Vortex for 2-3 seconds and allow to incubate at room temperature for at least 2min. 3. Insert sample and press "go" 4. Record reading and calculated numbers.

11.1.11

//I had trouble calculating the numbers from the fluorometer into fmoles. I contacted DR. Balza and explained the trouble I was having and he told me to abandon my research and pair up with Angela Blasezyk and to continue on with hers and to start morpholino injections.//

11.3.11

Angie and I pulled needles to prepare for morpholino injections. We pulled 6 fillament and 6 nonfillament needles and stored them in a covered petri dish until further use.

11.8.11 Angie continued with her miniprep while I was in class and also gathered fish in tanks for mating. When I returned from class we watched and waited for the fish to mate so we could inject the morpholinos, but they did not.

11.10.11 Angie ran the electrophorsis gel again with the new mini prep cut enzymes, and this time she did not see a band again

Morphilino 1. Angie and I got two tanks, filled them with a ratio of 2 female to 1 male 2. one tank had a divider which we removed after 15 minutes, these fish proceded to mate upon removal of divider. The other tank did not have a divider, these fish did not mate 4.we gathered the eggs using an egg net, and lined up the eggs single file along a glass slide in a petri dish filled with water 5. we then used a non filament needle, and set the pressure to about .8 8. then we injected TCX 43 morphilino into the embryos which were all at the one cell stage 9. we then removed the burst and dead embryos from the dish, and transferred the viable embryos into a beaker of egg water 10. placed in the incubator at 28.5 C

11.12.11

Angie returned to the lab and found all of our embryos were dead so she disposed of them.

Angie Continued with the mini-prep while I was out of town for Thanksgiving. The following is copied form her lab notebook. NOTE: "I" refers to Angie.

11/21/11

Subcloning the CNS into an expression vector using LR clonase 1. I decided to continue with the LR clonase reaction using the mini prep from 11/3 which produced one faint band 2. added .5ul of entry clone conataining CNS pME-CNS,.5ul pDes 1-02 A, 6ul of 1xTE buffer, and 2ul of LR clonase II plus enzyme 3.incubated at 25 C for 16hrs

11/22/11 1.added 1ul of Proteinase K and incubated at 37C for 10 mins 2.added 1ul of LR clonase reaction to a chilled tube 3. thawed top 10 E. coli cells and added 20ul of the cells 4. incubated on ice for 30 mins 5. heat shocked cells for 30seconds at 42 C 6. placed back on ice for 2 min 7. aseptically added 250ul of SOC meduim and shook at 225rpm for 1 hour 8. spread 100-200ul on ampicillin plates 9. incubated over night at 37 C

11/24/11 1.added bacteria from 4 colonies into seperate, 15ml tubes containing 5mL of LB broth, and 5ul of ampicillin 2.Let grow overnight

11/25/11 1.there was barely any bacteria, it didn't seem like I had enough to freeze and do a mini prep with 2.only 2 of the 4 samples had enough bacteria to work with and I did a mini prep 3.then I realized that I need to more bacteria to use to create a stock to inject 4. so I added bacteria from 4 other colonies, carefully selecting bactera from the outer edge of each colony and let grow overnight 5.tomorrow I will do the restriciton enzyme digest just to see what I get, as well as a new mini prep with the new bacteria

11/26/11 1.did mini prep with new bacteria samples

11/28/11 1. cut mini prep sample with EcoRV 2. ran 1% gel with 1kb ladder

11.29.11

Angie and I decided to try morpholino injections again today. Nick also joined us.

1. Angie got two tanks, filled them with a ratio of 2 female to 1 male with dividers between them. 2. The fish mated and 4.we gathered the eggs using an egg net, and lined up the eggs single file along a glass slide in a petri dish filled with water 5. we then used a non filament needle, and set the pressure to about 1.1 8. then we injected TCX 43 morphilino into the embryos. 9. We had trouble getting a good needle and damaged many of the embryos. Also, because we were already behind we were sort of rushing and only successfully injected 3 embryos. Even these three looked questionable. 9. we then transferred the viable embryos into a beaker of egg water and placed them in the incubator at 28.5 C
 * Because there was another group using the microinjection set up, we had to wait and some of our embryos looked like they might have started dividing into the 2 and/or 4 cell stage, although most of them were still 1 celled.

11.30.11

//Angie returned to the lab today and found all of our embryos dead.//