Bolda,+Emily


 * Genetics Lab**

9/1/11:**//Lab 1 Overview of transgenesis strategy, genomic DNA isolation, and bioinformatics//** //Lab Partners: Angie and Selciya//

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 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.

Continued....//9/7/11// 10. compared zebrafish and human sequence homology for my gene (MEF2c) on http://genome.lbl.gov/vista/index.shtml the above file shows the comparison. 11. Obtained the DNA sequence for each CNS on http://geneome.ucsc.edu as found: >danRer4_dna range=chr10:43461231-43461923 5'pad=0 3'pad=0 strand=+ repeatMasking=none GTGCCACCGTGTCACCCATATATATCAGAATTTTATATTACTTAAATAGA TTTTTATTGCTTCGTTTCAATTACACCATGTGTTTAGTTATGTAAATAAA GTGTATGTATTCTTATTTTATTGATTTAATTCATTTGCTATTCATGTTTT ATTATTTTTTAAATGCATTATTATTTTAAAAACACTTGATTGTTTGGCTT TTTCACCAAACATTTCCACTTAATGTACCCCTCAGACAGACCCTAGGGGC ACAGAAAGGGGTGTTGAATATAAACTCTCTGTATGTTCTCAA[GGAATCAT TTCTGTCACACTCCACTTGTGCGCTGATTTCAGATTGTGTGTTAGCATAA CAAAGACACAGAGCCTGTGAACTTGAGGGACCTTTCGACCCTGTGGCTCC GTCAATAGCAGCCCTCCTGCCTCTGTGCTTCGTGGAGAAAT]TGTTAATCA GCGTAATTTAATATAGTTTCTCGACTCATTCACACAGCTGCAGTCCGAAC ACAGTATTACTGTGCTTTACCATGGTGAAATGAGGAGATGGATGTCAATG TGGAGACCTGCTCTCAGACTAAAGCCTTAGATAAGAATGTGAACCTGGAA ATAAAAATGATTCTGTGATTAAGTAAATACCCATCTGATATATATATATA TATATATATATATATTTATTTATTTATTTATTTATTTATTTAT above is a picture of the chosen area around the CNS and below is a picture of only the CNS area.

Continued...//9/8/11// 12. With this information could plug into http://frodo.wi.mit.edu/primer3/ to determine the forward and reverse primer, Tm, and sequence size as seen in the document below. (In case it doesn't open I inserted the necessary information as well). Forward:GCACAGAAAGGGGTGTTGAA Tm:61.08 Product size:248 Reverse:GGACTGCAGCTGTGTGAATG Tm:60.47 13. Checked my primers via In silico PCR found on http://geneome.ucsc.edu which generated the charts below: GCACAGAAAGGGGTGTTGAAtataaactctctgtatgttctcaaggaatc atttctgtcacactccacttgtgcgctgatttcagattgtgtgttagcat aacaaagacacagagcctgtgaacttgagggacctttcgaccctgtggct ccgtcaatagcagccctcctgcctctgtgcttcgtggagaaattgttaat cagcgtaatttaatatagtttctcgactCATTCACACAGCTGCAGTCC ||  ||   ||   ||
 * || **UCSC In-Silico PCR ** ||  ||||   ||
 * || > [|chr10:43461479+43461726] 248bp GCACAGAAAGGGGTGTTGAA GGACTGCAGCTGTGTGAATG


 * || **Primer Melting Temperatures ** ||  ||||   ||
 * || ** Forward: ** 61.1 C gcacagaaaggggtgttgaa


 * Reverse: ** 60.5 C ggactgcagctgtgtgaatg

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. ||  ||   ||   || The primers match up along with the product size from In silico PCR. good to go.

9/6/11:**//Lab 2 Genomic DNA characterization and PCR//** //Lab Partner: David//

1. Prepared a .5% agarose gel with 2.5g agarose in 50ml 1XTAE in Erlenmeyer Flask 2.Microwaved about 40 seconds and paused to stir to avoid bubbling/over boiling, made sure there were no chunks 3. Added 2.5microliters GelStar to flask, stirred and poured in into mold(placed comb in gel) to harden. 4. Once gel was hardened, removed comb and covered with 1XTAE buffer (~325ml) in the electrophoresis rig.wells on black negative side. 5. Loaded 5ul of the DNA ladder into the first lane (High-range DNA ladder) 6. Combined 5ul of my DNA solution with 1ul 6XTriTrack loading dye in the 2nd slot. I tried mixing it in a microtube but it did not work so put DNA in first then the dye(could be a possible reason my DNA did not show up in the gel under the light or there was no DNA or I needed more dye). I did this with daivd's DNA in the third lane as well and Dr. Balza's DNA in the fourth lane as a constant. 7. Electrophoresed the get at 100v for about 70minutes. 8. Turned off the rig and placed gel in the Kodak Image 4000MM to see it under the fluorescence and took a picture. Also placed it under the fluorescent light in the back room.

Based on the shot taken the only item that showed up was the DNA ladder. I would assume at least Dr. Balza's DNA would show so maybe more dye was needed or the DNA was small and ran through? But obviously something was fishy.

9/15/11: //**Lab 3 Amplification and isolation of the enhancer using PCR & agarose gel extraction**//

1. Found my primer, matched it up with the forward and reverse primers from my in-silico PCR 2. microfuged my primer tubes for a few seconds so that the oligonucleaotide primer would be at the bottom of the tube. 3. diluted the primers with biology grade water to make a 100um stock solution of each primer. -right primer: 257 uL biology grade water -left primer: 295 uL of biology grade water 4. created 10um primer working solution: -10 uL of the right/forward primer -10 uL of the left/reverse primer -80 uL of molecular biology grade water 5. vortex the solution for a few seconds and stored tubes in freezer

Continued...//9/16/11// 6. Prepared master mix of standard Accurprime Taq Polymerse Reaction (for four 25uL PCR reactions in micro test tube) - 105 uL molecular biology grade water - 12.5 uL 10X Accuprime polymerse buffer II (contains MgCl2 & dNTPs) - 2.5 uL Genomic DNA in TE (used Dr. Balzas DNA) - 2.5 uL Primer mixing solution - 2.5 uL Accuprime Taq polymerse 7. placed 25 uL of the Accurprime Taq Polymerse Reaction in 4 .2ml PCR tubes and placed in Bio-Rad thermocyler 8. Put the tubes in slot A, B, C, D A : 63 degrees Celsius B: 62.3 degrees Celsius C: 60.9 degrees Celsius D: 58.7 degrees Celsius this was based of the Tm of 61.1 degrees Celsius for the forward primer and 60.5 degrees Celsius for the reverse primer 9. after the machine was done running, placed tubes in freezer. 10. Prepared a 1.5% agarose gel -.75g agarose -50ml 1XTAE 11. heated in microwave till dissolved and added 2.5uL GelStar 12. Poured gel n placed in comb, let solidify and stored in labeled ziploc bag.

Continued...//9/20/11// 13. Filled the electrophoresis with 1X TAE buffer until 1.5% gel was completely covered. 14. Loaded 5ul of the Generuler DNA 1KB DNA ladder into the first lane of my gel 15. Combined 5ul of my DNA solution for Tm from A, B, C, D and added 1ul of 6x TrikTrack loading dyes into lanes 2, 3, 4, 5. 16. Gel ran at 110v for about an hour. 17. Put in Kodak Image station and did not work, tried other light source in s203A and the picture did not turn out either. Possible Primer Dimers.

Continued...9/25/11 14. Made 2 gels of 1.5% agarose to set up for running of PCR. 15. Created a second Standard Accuprime Taq Polymerse Reaction with a mass stock solution of X5. This time I decreased the temperature a little bit to see if there would be any difference. A: 62 degrees Celsius B: 61.8 degrees Celsius C. 61.4 degrees Celsius D. 60.6 degrees Celsius

Continues...9/26/11 16. Set up the electrophoresis with 1X TAE buffer to cover my 1.5% agarose gel. 17. Loaded 5ul of the Generuler DNA 1KB DNA ladder into the first lane of my gel 18. Combined 5ul of my DNA solution for Tm (from the latest PCR product made on the 25th)A, B, C, D and added 1ul of 6x TrikTrack loading dyes into lanes 2, 3, 4, 5. 19. Ran another 1.5% gel the same way except with a 100bp DNA ladder and the original PCR product i made because I checked on how big my product was (248) so it may be easier to see if I have something with this ladder instead. 20. Gels ran at 110v for about an hour.

The picture below is the second time I ran PCR (made standard accuprime taq polymerse reaction) and It is with the 1KB DNA ladder in the 1.5% agarose gel:

21. took a clean scalpel and extracted my DNA from the gel and placed it in a microfuge tube (weighed the gel to mass) and stored in freezer.

Continues...9/27/11 22. Add 3 volumes (2100 uL for tube C) of Buffer QX1 DNA binding solution to 1 volume of gel (700uL). (1590uL Buffer for tube D which was 530uL for 1 volume of gel). Incubated 5 mins at 55 degrees Celsius. 23. Resuspended Qiaex II silica gel suspension by vortexing for 30secs. Added 10 ul of it to tube C and tube D. 24. Incubated at 50 degrees Celsius for 10mins. Vortexed every 2 mins to keep Qiaex II in suspension. 25. Centrifuged the sample for 30secs and disgarded the supernatent with pipet. 26. Wash pellet with 500uL of wash buffer QX1, resuspend by vortexing, centrifuged, removed supernatent with pipet. 27. Washed pellet twice with 500uL of wash buffer PE, resuspend by vortexing, centrifuged, removed supernatent. 28. Air-dried pellet for 10ish minutes 29. Resuspend pellet in 30uL of sterile TE buffer and incubate tube at 55 degrees for 5 mins, spin tube and removed supernatent into new microfuge tubes (tube C and D) 30. Stored DNA in freezer.

9/27/11: **Lab 4: Direct cloning of the CNS amplicon into an Entry vector using BP Clonase** 1. Characterization of the PCR product (CNS amplicon) quantity by fluormetric analysis 2. used the Broad Range Quant-iT Assay Kit and calibrated the Qubit fluorometer as directed in the Lab 4 Protocol. 3. Quantification of unknown DNA concentration by adding 2uL of purified PCR product to 198uL of Quant-IT BR working solution, vortexed and inserted into fluorometer: Tube C: .0179ug/mL (1.79ng/mol) Tube D: .0230ug/mL (2.3ng/mol)

Continued..9/28/11: Cloning CNS amplicon into entry vector using BP clonase 1. added my 7.5uL PCR product from the above result (discovered later was supposed to be my purified DNA) to microfuge tube 2. added MD-01 to tube (.5uL) 3. added 2uL of BP clonase II enzyme to tube 4. vortexed and incubated at 25 degrees Celsius for about 1hr 45mins 5. Added 1 uL Proteinase K solution and incubated at 37 degrees Celsius for 10mins 6.Stored tube in freezer

Continued...10/3/11: Transformation of BP clonase product into TOP10 chemically competent E. Coli 1. Added 1uL of BP clonase reaction to microfuge tube and placed in a bucked I filled with crushed ice. 2. Thawed a vial of TOP 10 E.coli on ice and flicked side of tube before adding 20uL of cells to my tube D. 3.Incubated on ice for 30mins 4. Heatshocked my tube with E.coli for 30secs at 42 degrees C 5. placed back on ice for 2 minutes 6. added 250 SOC medium to tube and placed on shaker for 1 hour 7. Set out Kana plate, sterilized glass spreader with Etoh and burned off over fire, allowed to cool. Centrifuged tube, took off SOC medium and placed on 250uL new SOC medium, flicked and triggerated cells with pipet, then placed on plate. Spread with glass rod. 8. Placed plates in the warm room, inverted for the night.

Continued...10/4/11 1. Had no colonies, redid transformation process with 2 changes and 1 addition: -placed SOC medium in warm room during shaking to prevent cold shock to cells when replacing the SOC medium -placed Kana plates in warm room during shaking to heat up plates -also used my tube C of BP clonase as well as my tube D

Continued...10/6/11 1. Still had no colonies, discovered used the DNA product from fluorometric anaylsis 2. Redid my BP clonase with purified PCR product for both tubes C and D 3. Stored in freezer

Continued...10/10/11 1.Redid transformation process with new BP clonase product. Placed in heat room overnight.

Continued...10/11/11 1. Got no colonies on plates again. Will have to test DNA in gel or run PCR again if DNA is no good.

Continued...10/13/11 1. Ran the purified DNA through a gel. got no DNA so would have to rerun the gel with the PCR product and redo the DNA gel extraction step to get better DNA product.

Continued...10/18/11 1. Ran PCR product through a gel (used the second round of PCR product made) and with the 100bp ladder and my product as seen in the picture below:

Continued...10/21/11 1. Redid the gel extraction with all the lanes shown in the picture. weighted the microfuge tube and then weighed the gel inside to determine the volume: A .13g B .12g C .12g D .12g 2. Add 3 volumes (390 uL for tube A) of Buffer QX1 DNA binding solution to 1 volume of gel (130uL). (360uL Buffer for tube B which was 120uL for 1 volume of gel; 360uL Buffer for tube C to 120uL gel; 360uL Buffer for tube D to 120uL gel). Incubated 5 mins at 55 degrees Celsius. 3. Resuspended Qiaex II silica gel suspension by vortexing for 30secs. Added 10 ul of it to tube C and tube D. 4. Incubated at 50 degrees Celsius for 10mins. Vortexed every 2 mins to keep Qiaex II in suspension. 5. Centrifuged the sample for 30secs and disgarded the supernatent with pipet. 6. Wash pellet with 500uL of wash buffer QX1, resuspend by vortexing, centrifuged, removed supernatent with pipet. 7. Washed pellet twice with 500uL of wash buffer PE, resuspend by vortexing, centrifuged, removed supernatent. 8. Air-dried pellet for 10ish minutes 9. Resuspend pellet in 30uL of sterile TE buffer and incubate tube at 55 degrees for 5 mins, spin tube and removed supernatent into new microfuge tubes (tube A,B, C and D) 10. Stored DNA in freezer.

Continued...10/23/11 1. Ran all the DNA through a gel to make sure that I had something after the extraction. All the DNA showed up but the DNA from A and B were the most abundant so I only continued with using tubes A and B for the next steps.

Continued...10/25/11: Characterization of the PCR product (CNS amplicon) quantity by fluormetric analysis 1. used the Broad Range Quant-iT Assay Kit and calibrated the Qubit fluorometer as directed in the Lab 4 Protocol. 2. Quantification of unknown DNA concentration by adding 2uL of purified PCR product to 198uL of Quant-IT BR working solution, vortexed and inserted into fluorometer: Tube A) .0389 ug/mL Tube B) .0430 ug/mL 3. Calculated the concentration to ng DNA: Tube A) 20.05 ng DNA Tube B) 22.16 ng DNA

Continued...10/27/11: Cloning of the CNS amplicon (PCR product) into an entry vector using BP clonase 1. added my 7.5uL PCR product from (tubes A and B to two separate microfuge tube) 2. added MD-01 to tube (.5uL) 3. added 2uL of BP clonase II enzyme to tube 4. vortexed and incubated at 25 degrees Celsius for about 1hr 45mins 5. Added 1 uL Proteinase K solution and incubated at 37 degrees Celsius for 10mins 6.Stored tubes in freezer.

Continued...10/30/11: Transformation of BP Clonase into TOP10 chemically competent E. Coli 1. Followed the steps in the protocol for this step in lab 4 with only a few changes. (used tubes A and B) 2. since there were no more kana plates, let the cells with my DNA (in the tube) thaw about 35-40mins on the ice before heat shocking. 3. Left the tubes on the shaker for 1 hour 45 mins...while i waited for the plates to be poured and cooled enough to plate my cells, my microfuge tube cells sat for about 30mins. 4. Then I did NOT centrifuge the cells down this time and replace with new soc medium. So I directly was able to plate the cells and put them in the warm room to grow.

10/31/11: **Lab 5: Subcloning the CNS into an expression vector using LR clonase** 1. Got bacteria to grow on plates to work!! whoo hoo! 2. Since David Bartels and Rebecca Schulz were working together with Davids gene (which was the same as mine) and they could not get colonies, we all grouped together and (used my colonies) for the rest of the lab. We decided that all of us do the lab sections until one person cant get it to work so we would have a better chance of not redoing parts of the lab and being successful! We also would have like 3 times as much for almost all the steps so we wouldn't have to go back so far.

Continued...11/1/11: Selection of colonies which may contain the entry vector with your zebrafish CNS insert 1. Carefully transferred four colonies to separate conical tubes ( I did 4) David and Becca also made tubes as well that had 5mL LB media with kanamycin (5uL). 2. I put mine in the warm room on the shaker without completely closing the top so air could get in. This was in overnight

Continued..11/2/11 1. My bacteria was not very cloudy so I left mine in the shaker another night and preserved a portion of the bacteria still anyways. 2. combined 1.7mL of E. Coli in Lb media with .3mL of 80% Glycerol to yield a 20% glycerol storgae solution in a 2mL Cryopreservation tube. Vortexed shortly and stored the tube in the -80% frig. (David and becca also did the same with theirs)

Continued...11/3/11 1. My bacteria grew more so I made another preservation tube as stated in the step above.

Continued...11/7/11: Isolation of the entry clone plasmid DNA containing your sebrafish CNS by plasmid miniprep 1. Used the QIAprep Spin Plasmid miniprep kit (did two microfuge tubes total for this step) 2. Centrifuged about 1.5mL of the E. coli grown in our conical tubes 3. Took off the supernatant and resuspended the pelleted cells in 250uL of Buffer P1. vortexed till there were no clumps. 4. Added 250 uL of the SDS-alkaline lysis solution (Buffer 2) and mixed by inverting the tube about 6 times. 5. added 350uL of the neutralization solution (Buffer N3) and mix immediately by inverting 6 times. 6. centrifuged for 8-10mins 7. Transferred supernatant with plasmid DNA to the QIAprep spin column avoiding the white precipitate. 8. Centrifuged for close to a minute. Discarded the flow through and placed the column back into the same collection tube 9. Added 500uL of the binding solution (Buffer PB). Centrifuged for close to a minute and discarded the flow through. placed column back into the same collection 10. Added 750uL of the ethanol-containing wash solution (Buffer PE). centrifuged for almost a minute. Discarded the flow through and placed the column back in the collection tube. 11. Centrifuged additional minute to remove residual wash. 12. Transfer the QIAprep to a clean microfuge tube (cut of the lid) and eluted the column with 50uL elution buffer (buffer EB) pipetted onto the center of the column membrane. Allowed to elution buffer to stand on the column one minute and centrifuged for 1 minute. 13. Discarded the column and sotred the eluted plasmid at -20 degrees. (David and Becca did the same)

Continued...11/8/11: Confirmed the correct size of cloned CNS insert with a restriction enzyme digest 1. We pulled up the NEBcutter 2.0 software ( http://tools.neb.com/NEBcutter2/index.php) to see if the restriction enzymes Apal and EcoRV would cut the CNS in any other places. 2. It showed that it would not be cut anywhere else so with these enzymes we should see two bands in our gel. (2330bp and about a little over 300bp) 3. We combined the following in a microfuge tube to prep for the gel: (I did this for both of my miniprep tubes) H2O-18uL 10x NEB buffer #4- 2.5uL 10x BSA- 2.5uL Plasmid DNA- 1.0uL Apal enzyme- 1.0uL Incubated at 25 degress for 1 hour 4. Added 2uL of EcoRV and incubated for another hour at 37 degrees. 5. When finished we put in the freezer to store.

Continued...11/11/11 1. We ran a (1.5%)gel with the 1kb ladder, the 100bp ladder, both my tubes of plasmid DNA with restriction enzymes, and davids one plasmid DNA with restriction enzyme, and two rows with just 5uL of my plasmid DNA, and one row with 5uL of Davids plasmid DNA. 2. We saw all the plasmid DNA in the gel, both ladders, but we only one clear band and a very faint band in all of our plasmid DNa with restriction enzymes. (my plasmid DNA #1 tube had a little more DNA than my #2 tube) 3. This meant we would need to redo the amount of DNA put in with our restriction enzymes.

Continued...11/15/11 1. we redid the amount by changing the H2O to only 14uL and the DNA to 5uL for our restriction enzyme reaction. Otherwise we did the same for everything else. I only used my number 1 tube for this part then.

Continued...11/16/11 1. Ran our 1.5% gel again with the 1kb ladder, 100bp ladder, my rows of plasmid DNA with restirction enzymes, and davids 1 row with restriction enzymes. 2. Gel showed that there were two bands so we know it worked!

Continued...11/18/11 Subclone your CNS the destination vector containing a reporter gene using an LR reaction 1. Added .5uL of my CNS about .5uL of pDes1-01 (David did pDes1-02 and becca did both) and 1XTE buffer about 6.5uL 2. Vortexed LR Clonase II Plus enzyme mix for 2 sec (did it twice total). and added 2 uL to the components above. 3,incubated reaction at 25 degrees for about 16hours.

Continued...11/19/11 4. Took off the hot plate and added 1 uL of 2ug/ul Proteinase K solution and incubate at 37 degrees for 10 minutes 5. Put in freezer to store.

Continued...11/20/11 Transformation into chemically competent TOP10 E. Coli 1. Added 1uL LR clonase to microcentrifuge tube and placed on ice 2. Thawed vial TOP10 E.coli cells on wet ice and tap to mix before adding 20uL of cells to my tube 3. Incubated this mix on ice for 30minutes 4. Heat shock the cells for 30 seconds at 42 degrees 5. Place heat-shocked cells back into the ice for 2minutes 6. Added 250uL of SOC medium to the cells 7. put on shaker for an hour and 45 minutes 8. Let sit for about 10minutes and then spread 150uL on ampicillin plates. 9.Incubated overnight

Continued...11/21/11 1. Had colonies grow so chose four to place into four 15mL steril conical tubes (had 5mL LB media with 5uL ampicillin) 2. left lid on lose for air flow

Continued...11/22/11 1. David cryogenically preserved his E. Coli because my tubes did not grow enough yet and we didn't have time before thanksgiving to wait. (did grow when we got back but I continued with Davids bacteria) 2. so David preformed the isolation of the destination vector containing the CNS and reporter gene by plasmid miniprep. 3. He used the QIAprep Spin Plasmid miniprep kit as used in the lab previously. Found in the Lab 5 protocol. Did nothing different.

Continued...11/27/11 1. Becca ran a gel with EcoRV and confirmed that it worked (two bands) so we were ready to begin injections.

11/27/11**: Lab 6: Microinjection of transgenes into zebrafish embryos** 2. David and I began the Large Scale prep of plasmid DNA for microinjections: 3. we took his leftover bacteria in LB and took 50mL of this and 50uL of ampicillin and pit on the shaker for about 19hours

Continued...11/28/11 1.We centrifuged the conical tubes down and took off the supernatant. We parafilmed it and stored it in the freezer. 2. We made our needles with filament and non filamented ones using capillaries on the Narishige PC10 puller. We broke the tips to make any needles blunter. 3. We obtained embryos and put on a petri dish with a microscope slide taped to the side. We lined up the embryos on this for injections. 4. We had a both mCherry and GFP to chose from for our cocktail mix. 5. We dilute the DNA: 10uL plasmid DNA 5uL transposase RNA? 345uL sterile dH20 40uL 10x zebrafish injection cocktail 6. We placed the cocktail on a petri dish and sucked it up in our needle and began injecting WT embryos. 7. Once completed were rinsed into a petri dish with 1xegg water

Continued...11/29/11 1. Possible transgenetic fish! 2. We looked at it with Dr. Balza and he advised that we pull off the membrane and take pictures of our fish with control fish. Which we did and then placed our buddy into the petri dish with fresh egg water.

Continued...11/30/11 1. We showed Dr. Balza our pictures with the two glowing dots. Official trangenetic fish! 2. We continued to monitor with new water and see that he was alive

Continued...12/3/11 1. Injected INS:mCherry embryos with our GFP cocktail mix. and placed in petri dishes with 1xegg water

Continued...12/4/11 1. Checked on the fish and took out any of the dead ones. Saw nothing glowing under the light. 2. Checked on transgene fish and took pictures. still alive

Continued...12/5/11 1. David checked on INS:mCherry embryos and took pictures. Possible glowing but unsure.