Bartels,+David

Zebrafish genetics

September 1, 2011

Lab 1: Overview of transgenesis strategy, genomic DNA isolation, and bioinformatics

1. Anesthetized one zebrafish in 200mg/L tricaine. 2. Sacrificed fish with a scalpel, cut off tail and head. 3. Used liquid nitrogen to snap freeze fish. 4. Ground frozen fish into a powder with a mortar and pestle. 5. Added powdered fish tissue to 7.5 ml lysis buffer, shook to submerge tissue. 6. Allowed to incubate for 60 minutes on a rocking platform. 7. Used Shepherd's crook to gather DNA in lysis/EtOH solution. 8. Transferred DNA to a tube containing 1ml of TE.

Vista Browser Screen Shot: This picture shows the mef2c gene. The areas of CNS that we are interested in are in red and can be seen before the actual gene as well as after.

CNS: >danRer4_dna range=chr10:43475024-43475596 5'pad=200 3'pad=200 strand=+ repeatMasking=none CGCTTAAGCACAATTTGGGAAATATTTGAAAGAAATCACAGGAGGGTGAA TGATTCCGACTACAACTGTATAGATATTGAGTGTCCTGTGAGAATCCTGT GAGTCTCCCCCTTCACTGTGTCTCACCCAGCGGTTCCCATCCGCTTCTGA AGCCCTCAACAGCCCAAACCGAGAGCCCGAACCCCCCAGCCCACCCTAAT [ GAGGGTCCCGGGGACAATACGGGCATTGTTTGGCCCCTCAGCTCAAGAAC AATGGATGTTTACCCAAGCAGTCGTCCGACCCAAACTCTGCCGTTGATTA CTGTTGGGTCTCCTGATTAACTCTGCTCCTCGACCCAGCACAATGGAGCC TTTGACTGCACGACCCGACAAAA ]AAAAAGCCCCCTCCAATTTGTCCTCTT TCTCTCCATATACACTCGTAACACTGAACTCCATTATGGGACCCGAGAGA GAGCAGCGTCTCTGAAACATCCACACACACTTCAGTTGTGCTGATACCTG TTAAAACCGCAGCTGAAATCAGGCCTTTGTGCACTGAAATACGCACACAA GCGCTTCATAAAGGGTCGATGCA

Left Primer: CTTCTGAAGCCCTCAACAGC tm: 60.13 degrees C Right Primer: AAATTGGAGGGGGCTTTTT tm: 59.76 degrees C

UCSC In-Silico PCR ||
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[]
 * || Rating || Sequence || Position || Length(bp) || Tm C || GC% ||
 * Sense ||  || CTTCTGAAGCCCTCAACAGC || +144 || 20 || 60.13 degrees C || 55.00 ||
 * Antisense ||  || AAATTGGAGGGGGCTTTTT || -392 || 19 || 59.76 degrees C || 42.11 ||
 * Product ||  ||   ||   || 573 ||   ||   ||

Lab 2: 1. Prepared a 0.5% agarose gel (.25g of agarose in 50 ml of 1XTAE, heated in microwave for 30sec-1min) 2. Added GelStar DNA stain to 0.5x concentration. (2.5 microliters per 50ml of gel) 3. Poured agarose into gel mold, inserted comb. 4. Removed comb, covered with 1x TAE buffer in electrophoresis rig. 5. Loaded 5 microliters of DNA ladder into first lane. 6. Combined 5 microliters of DNA solution with 1 microliter of 6xTriTrack loading dye, loaded into lane 2. 7. Started electrophoresis (red=positive and black =negative) at 100 volts for 30-60 minutes. 8. Photographed gel.

Electrophoresis gel photograph: 9/15/11: //**Lab 3 Amplification and isolation of the enhancer using PCR & agarose gel extraction**// 1. Found primer, matched it up with the forward and reverse primers from in-silico PCR 2. microfuged 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: 291 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: C: D: 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 in placed in comb, let solidify and stored in labeled ziploc bag.

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 fluorometric 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 7.5uL of 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 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 tube. 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: -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

Continued...10/6/11 1. Still had no colonies 2. Redid my BP clonase 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 DNA, tried to extract the gel containing DNA better this time.

Continued...10/21/11 1. Weighted the microfuge tube and then weighed the gel inside to determine the volume. 2. Add 3 volumes of Buffer QX1 DNA binding solution to 1 volume of 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 tubes. 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. 10. Stored DNA in freezer.

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. 3. Calculated the concentration to 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 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. 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 i****nto an expression vector using LR clonase** 1. Got bacteria to grow on plates to work 2. Since Rebecca Schulz and I were working together with my gene (which was the same as Emily's) and we could not get colonies, we all grouped together and (used Emily's colonies) for the rest of the lab. We decided that we would all do the lab sections until one person was not able to 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 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, also made tubes as well that had 5mL LB media with kanamycin (5uL). 2. Put tubes in the warm room on the shaker without completely closing the top so air could get in. Then left overnight.

Continued..11/2/11 1. Bacteria was cloudy so I 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. 2. Vortexed shortly and stored the tube in the -80% frig.

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.

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 ( []) 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: 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. 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 of Emily's plasmid DNA with restriction enzymes, and my one plasmid DNA with restriction enzyme, and two rows with just 5uL of Emily's plasmid DNA, and one row with 5uL of my 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. 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. Emily only used her number 1 tube for this part.

Continued...11/16/11 1. Ran our 1.5% gel again with the 1kb ladder, 100bp ladder, Emily's rows of plasmid DNA with restirction enzymes, and my 1 row with restriction enzymes. 2. Gel showed that there were two bands so we know it worked. (Band that corresponds to our CNS was faint because there is less DNA there for the TriTrack to attach to, and therefore, glow)

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 pDes1-02 (Emily did pDes1-01 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.