Haupt,+Katie

Zebrafish genetics

__Lab 1:__ __Overview of transgenesis strategy, genomic DNA isolation, and bio informatics__
 * Isolation of zebrafish genomic DNA
 * five specemins of zebra fish were obtained and anesthetized using 200mg/L tricaine until no longer responsive
 * the fish were then decapitated and cut up into small sections excluding fins and tail fin
 * liquid nitrogen was then used to snap freeze the tissue, and was pulverized into a powder with a mortar and pestle
 * after the liquid nitrogen evaporated, the fish powder was added slowly and carefully to 7.5ml lysis buffer in a 50ml polypropylene conical tube and after all the material was in solution, it was incubated at room temperature on a rocking platform for 45 min.
 * the lysis buffer solution was then carefully transfered into a seperate 50ml conical tube containing 18ml of 190 proof EtOH
 * the DNA was then recovered by slowly stirring the interface between the lower aqueous lysis solution and the upper EtOH with a Shepherd's crook (previously made using pipettes)
 * crooks then removed allowing as much ethanol to drain away causing the DNA to shrink into a tightly packed, dehydrated mass; the DNA was then washed in a fresh conical tube containing 5ml EtOH
 * as much EtOH was then removed as was possible but not allowing the DNA to dry completely
 * Each pipette was then transfered to a fresh polypropylene tube containing 1ml of TE (pH 8.0), allowing the DNA to rehydrate overnight at 4 degrees Celcius
 * after rehydration was completed, the DNA solution was transfered to a microfuge tube for storage at 4 degrees Celcius
 * Identification of conserved non-coding (putative enhancer) sequences
 * used Vista browser to compare human and zebra fish genomes
 * trial 1
 * DNA sequence of cns on my gene (T-Box transcription factor 5)
 * Chr5:75,673,325-75,674,923
 * Primer design
 * forward primer: CACGCACGCCATATTTACAC
 * reverse primer: ATCACCTTTGGCCTCGTGAG
 * trial 2
 * DNA sequence of cns on my gene (T-Box transcription factor 5)
 * Chr5
 * primer design
 * forward primer:
 * reverse primer:
 * trial 3
 * DNA sequence of cns on gene (CEI-IRX1)
 * chr16:612,021-613,842
 * primer design:
 * forward primer: GCAATTTTGCCAATGGTTTCC
 * temperarrue: 63.6 deg. C
 * reverse primer: CCAAAGCCTTCAAATGCCTCGC
 * temperature: 69.0 deg. C
 * DNA sequence of cns on gene (FAF1)
 * chr3:6,608,290-6,610,105
 * chr8:17,283,370-17,285,185
 * primer design:
 * forward primer: TCTGTGGCATTCTCTTCGGCC
 * temperature: 67.0 deg. C
 * reverse primer: TGGCATCTCGCACACAAAAGC
 * temperature: 66.8 deg. C

__Lab 2: Genomic DNA characterization__
 * Characterization of genomic DNA quality by agarose gel electrophoresis
 * a 0.5% agarose gel was prepared by heating 0.259g agarose in 50ml 1xTAE in the microwave for about 2.5 min. in a Ehrlenmeyer flask
 * 2.5 microliters of GelStar Necleic Acid Stain was added to the dissolved agarose
 * the dissolved agarose was then poured into a gel mold and allowed to solidify after placing a comb in one end, this tok about 10 minutes or so
 * after agarose gel was solid, the comb was removed and the gel was placed in a electrophoresis rig and covered with 1x TAE buffer using minimal amount of the buffer to just cover the gel with the wells of the agarose gel near the negative (black) electrode and the base of the gel near the positive (red) electrode
 * contents of the lanes:
 * lane one: 5 microliters of DNA ladder (high range DNA ladder was used)
 * lane two: 5 microliters control DNA combined with 1 microliter 6x TriTrac loading dye
 * lane three: 5 microliters of sample zebra DNA combined with 1 microliter 6x TriTrac loading dye
 * gel was electrophoresed at ~100v for about two and a half hours
 * gel was then imaged using the Kodac Image station 4000MM[[image:lab_2_gel.png]]

__Lab 3: Proximal promoter PCR amplification & agarose gel extraction__
 * Oligonucleotide primer dilution
 * briefly spin the microfuge tube containing oligonucleotide primer to ensure that it is at the bottom of the tube
 * ~30 seconds
 * dilute oligonucleotide primers in molecular biology grade water to create a 100uM stock solution of each primer, briefly vortex the solution
 * create a 10uM primer working solution by adding 10uL of the sense forward primer and 10uL of the antisense reverse primer to 80uL of molecular biology grade water; briefly vortex the solution
 * label tube well
 * store all tubes @ -20 deg. C
 * Polymerase chain reaction (PCR)
 * prepare the folowing 25uL PCR reacions in each of four thin-wall 0.2mL PCR tubes
 * Standard Accuprime Taq Polymerase Reaction
 * 21 uL molecular biology grade water
 * 2.5 uL 10x Accuprime polymerase buffer II
 * 0.5 uL Genomic DNA in TE
 * 0.5 uL Primer mix (working solution)
 * 0.5 uL Accuprime Taq polymerase


 * Trial 1
 * Row A: 60.0 deg. C
 * Row B: 59.8 deg. C
 * Row C: 59.2 deg. C
 * Row D: 58.3 deg. C
 * Row E: 57.0 deg. C
 * Row F: 56.1 deg. C
 * Row G:55.5 deg. C
 * Row H: 55.0 deg. C
 * Trial 2
 * Row A: 59.0.0 deg. C
 * Row B: 58.5 deg. C
 * Row C: 57.5 deg. C
 * Row D: 55.8 deg. C
 * Row E: 53.5 deg. C
 * Row F: 51.9 deg. C
 * Row G: 50.7 deg. C
 * Row H: 50.0 deg. C
 * Trial 3
 * Row A: 58.0 deg. C
 * Row B: 57.6 deg. C
 * Row C: 56.6 deg. C
 * Row D: 55.2 deg. C
 * Row E: 53.1 deg. C
 * Row F: 51.7 deg. C
 * Row G:50.7deg. C
 * Row H: 50.0 deg. C
 * Agarose Gel purification of PCR amplicons
 * Trial 1
 * used a 1.5% agarose concentrated gel (0.75g agarose per 50mL 1X TAE buffer)
 * Lane 1: DNA Ladder
 * Lane 2: A
 * Lane 3: B
 * Lane 4: C
 * Lane 5: D
 * Lane 6: E
 * Lane 7: F
 * Lane 8: G
 * Lane 9: H
 * Results:[[image:gel#1.png]]
 * Trial 2
 * used a 1.5% agarose concentrated gel (0.75g agarose per 50mL 1X TAE buffer)
 * Lane 1: DNA Ladder
 * Lane 2: D
 * Lane 3: E
 * Lane 4: F
 * Lane 5: G
 * Results: [[image:pcr2_gel1_kmh.png]]
 * Trial 3
 * used a 1.5% agarose concentrated gel (0.75g agarose per 50mL 1X TAE buffer)
 * Lane 1: DNA Ladder
 * Lane 2: B (FAF 1)
 * Lane 3: C (FAF 1)
 * Lane 4: D (FAF 1)
 * Lane 5: E (FAF 1)
 * Lane 6: A (CEI-IRX1)
 * Lane 7: B (CEI-IRX1)
 * Lane 8: C (CEI-IRX1)
 * Lane 9: D (CEI-IRX1)
 * Resutls: [[image:kmh_FAF1_&_CEI-IRX1.png]]
 * Amplicon extraction protocol
 * Cut out a slice of gel containing the amplicon band with a clean scalpel. Minimize the time of UV exposure as much as is practical, as this will cause mutations. minimize the size of the gel slice by trimming excess agarose.
 * Determine an approximate volume of gel slice by weight by weighing the gel slice in a 1.5mL plastic microfuge tube. Be sure to weigh tube before adding the gel slice, so that you can determine the mass of the gel by subtraction.
 * FAF1: 0.33g
 * CEI-IRX1: 0.30g
 * Add 3 volumes of Buffer QXI DNA binding solution to 1 volume of gel. Incubate 5 minutes at 55 deg. C to dissolve agarose.
 * Resuspend the Qiaex II silica gel suspension by vortexing for 30 sec. Add 10uL of the resuspended Qiaex II to the tube containing your gel slice.
 * Incubate at 50 deg. C for 10 min. to solubize the agarose and bind the DNA to the silica. Mix by vortexing every 2 min. to keep the Quiaex II in suspension. Be sure that the color fo the mixture is yellow! If it is purple, you must imediately add 10uL 3M sodium acetate, pH 5.0 to adjust the pH.
 * Centrifuge the sample for 30 sec. and carefully discard the supernatent with a pipet.
 * Wash the pellet with 500uL of wash buffer QX1. Resuspend by vortexing, pellet with a brief centrifugation, and remove the supernatant with a pipet. This step removes residual agarose.
 * Wash the pellet twice with 500uL of wash buffer PE. Resuspend by vortexing, pellet with a brief centrifugation, and remove the supernatent with a pipet. These steps remove residual salts.
 * Air-dry th pellet for 10-15 min. or until the pellet appears white. Do not overdry, as this will decrease elution efficiency.
 * Elute DNA into TE buffer (pH 8.0). Resuspend the pullet in a 30uL sterile TE buffer adn incubate the tube at 55 deg. C for 5 minutes. Spin the tube and remove the supernatent into a new microfuge tube, taking care to avoid the pellet.
 * Store the DNA in a well-labeled tube at -20 deg. C.

__Lab 4: Direct cloning of the CNS amplicon into an entry vector using BP clonase__
 * Cloning of the CNS amplicon (PCR product) into an entry vector using BP clonase
 * Add the following components to a 1.5mL mirocentrifuge tube at room temperature and mix:
 * attB PCR product, 7.5uL
 * pKONR 221 or MD-01, 0.5uL
 * BP Clonase II enzyme/buffer mix, 2uL
 * Vortex the 10uL briefly and incubate reaction at 25 deg. C (room temp) for 1-2 hours.
 * Add 1uL of 2ug/ul Proteinase K solution and incubate at 37 deg. C for 10 minutes.
 * Transform 1uL of the reaction into competent E. coli as described below or store the BP reaction at -20 deg. C.
 * Transformation of BP clonase reaction product into TOP-10 chemically competent E. coli
 * Add 1uL of the BP clonase reaction product to a microcentrifuge tube and place on ice to chill.
 * Thaw a vial of TOP10 E. coli cells on wet ice (one vial for two transformations).
 * When cells are thawed, mix cells by tapping gently (DO NOT VORTEX OR PIPETT AS COMPETENT CELLS ARE FRAGILE). Add 20uL of cells to each chilled microcentrifuge tube containing DNA.
 * Incubate the vial of cells + BP reaction product on ice for 30 minutes.
 * Transform the DNA into the cells by heat-shoking the cells for 30 seconds at 42 deg. C without shaking.
 * Place the heat-shocked cells back into the ice for two minutes.
 * Aseptically add 250uL of S.O.C. medium to the cells.
 * Shake at 225rpm (37 deg. C) for 1 hour.
 * Spread 100-200uL of hte transformed cells on a prewarmed kanamycin plate.
 * Incubate inverted plate(s) overnight at 37 deg. C.
 * The plate should yeild clonal colonies of TOP10 E. coli that contain the product of the BP﻿ clonase reaction. Wrap plates with parafilm and store at 4 deg. C until you are ready to grow the colonies up.

__Lab 5: Subcloning the CNS into an expression vector using LR clonase__
 * Selection of colinies which may contain the entry vector with your zebrafish CNS insert
 * Carefully transfer (using a fresh sterile toothpick or similar implement) four colonies to four seperate 15mL sterile conical tubes containing 5mL LB media with kanamycin. Allow to grow overnight in the warm-room shaker. Note that this works best when the cap is left loose to facilitate gas exchange. Ensure that the cap does not fall off completely by attaching the cap with a piece of tape.
 * Cryogenically preserve a portion of hte bacteria containing the product of your BP reaction (entry clone containing the CNS) as follows:
 * Combine 1.7mL of E coli in LB Media with 0.3mL of 80% Glycerol to yield a 20% glycerol storage solution in a 2mL Cryopreservation tube. Vortex briefly, carefully label the tube adn store at -80 deg. C in the box marked "Fall 2010 zebrafish trasgenesis bacterial stocks."
 * Isolation of the entry clone plasmid DNA containing your zebrafish CNS by plasmid miniprep