Blasezyk,+Angela

9/1/11 Angie Blasezyk Lab 1: Overview of transgenesis strategy, genomic DNA isolation, and bioinformatics Lab Partners: Emily Bolda, Selciya Lamech

1. Anesthtize zebrafish with dilluted tricane (200mg/ml) 2. Cut off fins and head 3. Snap froze in liquid nitrogen and made into a powder/slush 4. Added crushed fish to lysis buffer (7.5mL) in a conical tube and allowed to incubate for 45mins 5. Transfered lysis buffer into a new 50mL conical tube of 18mL of 190 proof EtOH 6. Recovered DNA by swirling a plastic stick in the tube, rinsed in EtOH, and added DNA to new tube and refridgerated

DNA sequence for CNS 1 (brackets) 200bp then CNS then 200bp

chr10:43,500,166-43,500,255 (Angie)

>danRer4_dna range=chr12:15991562-15992302 5'pad=200 3'pad=200 strand=+ repeatMasking=none

TAGGTAGAAAATGTATCTTTCAGAAGGCACGTCTCTAGGAAAATCAATCC TCACAATCACATTTACAGAAATGTAGATCAAATCTTTCTCAGAAAACACT CTAACCAGCCAAAATATTTAAAGCCAGCTTACACCTGTGCAAAAAACAAC AACAACAACAACAGCACTTCTTTGACCCTTTACACAGCGAGTTTCAAGAG [TAATCAGATAAAGCTTGCAAAATAGAAAGAGAAGGAGGTGAGAGAAGCTA GGTGGCACTTACCCTGACAGTGCTGGGCCGGGGGGGTATTTAGCCACTTG GTGATGGTGTACACCTCACTGGTTTGTGGTGTACTTATGTAGTTTCCATC TCTGCTCACCACACCCTCTAACATCCCAAACAAGGAGCCTGGGCTGCACT CGGATCGGTAGCCTCACTCCACTAAAGCCTTATAGGGATGACTGGGAGAA AACACTCCAATCCGGGCAGCACCAGGGCGGCCTCTTGCCTCCTTACTCTG GGCATTCCAATGGACCTTTTCCTTTTTTCTCCTCAGAAATTGTGGGGACT] TCACTATGCACGAGAATAAACACAGGGCTGGGCCTGATCTGCTACACAGA CGCTCACCACGGCCCACACCTGAGAAGGAATGGCAAAGACATTTGGTCAC TTAAGTACATAGGTCACACGAGTTTAAAAGTTTAAACTCCCAAGCTGTAC TGTAAAAACAAAGCAAAAAGTAAATAAAATAAAATCTTATG

DNA sequence for CNS 2 chr10:43,474,982-43,475,628 DAVID

>danRer4_dna range=chr10:43474982-43475628 5'pad=0 3'pad=0 strand=+ repeatMasking=none ATGCAAAAACATCATAGGAAATATTGTGAAAACCCCATTGCTCGCTTAAG CACAATTTGGGAAATATTTGAAAGAAATCACAGGAGGGTGAATGATTCCG ACTACAACTGTATAGATATTGAGTGTCCTGTGAGAATCCTGTGAGTCTCC CCCTTCACTGTGTCTCACCCAGCGGTTCCCATCCGCTTCTGAAGCCCTCA ACAGCCCAAACCGAGAGCCCGAACCCCCCAGCCCACCCTAATGAGGGTCC CGGGGACAATACGGGCATTGTTTGGCCCCTCAGCTCAAGAACAATGGATG TTTACCCAAGCAGTCGTCCGACCCAAACTCTGCCGTTGATTACTGTTGGG TCTCCTGATTAACTCTGCTCCTCGACCCAGCACAATGGAGCCTTTGACTG CACGACCCGACAAAAAAAAAGCCCCCTCCAATTTGTCCTCTTTCTCTCCA TATACACTCGTAACACTGAACTCCATTATGGGACCCGAGAGAGAGCAGCG TCTCTGAAACATCCACACACACTTCAGTTGTGCTGATACCTGTTAAAACC GCAGCTGAAATCAGGCCTTTGTGCACTGAAATACGCACACAAGCGCTTCA TAAAGGGTCGATGCAGGACAAAATAATGCTAAATAAAATGCTTTTCT

DNA sequence for CNS 3 chr10:43,461,250-43,461,905 EMILY

>danRer4_dna range=chr10:43461250-43461905 5'pad=0 3'pad=0 strand=+ repeatMasking=none ATATATCAGAATTTTATATTACTTAAATAGATTTTTATTGCTTCGTTTCA ATTACACCATGTGTTTAGTTATGTAAATAAAGTGTATGTATTCTTATTTT ATTGATTTAATTCATTTGCTATTCATGTTTTATTATTTTTTAAATGCATT ATTATTTTAAAAACACTTGATTGTTTGGCTTTTTCACCAAACATTTCCAC TTAATGTACCCCTCAGACAGACCCTAGGGGCACAGAAAGGGGTGTTGAAT ATAAACTCTCTGTATGTTCTCAAGGAATCATTTCTGTCACACTCCACTTG TGCGCTGATTTCAGATTGTGTGTTAGCATAACAAAGACACAGAGCCTGTG AACTTGAGGGACCTTTCGACCCTGTGGCTCCGTCAATAGCAGCCCTCCTG CCTCTGTGCTTCGTGGAGAAATTGTTAATCAGCGTAATTTAATATAGTTT CTCGACTCATTCACACAGCTGCAGTCCGAACACAGTATTACTGTGCTTTA CCATGGTGAAATGAGGAGATGGATGTCAATGTGGAGACCTGCTCTCAGAC TAAAGCCTTAGATAAGAATGTGAACCTGGAAATAAAAATGATTCTGTGAT TAAGTAAATACCCATCTGATATATATATATATATATATATATATATTTAT TTATTT

9/6/11 Angie Blasezyk Lab 2: Genomic DNA characterization & PCR

1. Made .5% agarose gel -.35g agarose - 50mL of 1x TAE -heated mixture until it boiled 2. Added 2.5 uL of GelStar which is a nucleic acid stain 3. Poured Gel and waited 10 minutes 4. Removed comb and filled apparatus with TAE buffer just enough to cover over the gel (to maximize rate of electrophoresis) 5. Loaded .5 uL of DNA ladder (high range DNA latter (10,171-48,502bp) 6 Then loaded a combination of control DNA (5ul) and zebra fish DNA (5ul) each mixed with 1ul of TriTrack loading dye in the wells on the negative end of the terminal 7. Set the voltage to 100v and let run for 35mins 8. Examined gel under UV light 9. Only the control DNA and ladder spread through the gel, so I will need to find a different source of DNA

9/8/11

Designing Primer

comparing human genome with zebrafish andother vertibrates to determine important CNS. Important ones will show up in all the animals. [|http://pipeline.lbl.gov/tbrowser/tbrowser/?pos=chr5:88049815-88215058&base=hg18&run=80&genes=refseq5#&base=94&run=80&run=6&pos=chr5:87884569-88215056&genes=refseq5&indx=1&cutoff=50]

DNA CNS 1 above

pasted it onto following website to acquire primer sequence

[]

SEQUENCE SIZE: 741 INCLUDED REGION SIZE: 741


 * || Sequence || Position || Length (bp) || Tm C || GC% ||
 * Left primer || CCCTTTACACAGCGAGTTTCA || 176 || 21 || 60.29 || 47.62 ||
 * Right primer || TGTGTTTATTCTCGTGCATAGTGA || 574 || 24 || 59.71 || 37.5 ||

1 TAGGTAGAAAATGTATCTTTCAGAAGGCACGTCTCTAGGAAAATCAATCCTCACAATCAC 61 ATTTACAGAAATGTAGATCAAATCTTTCTCAGAAAACACTCTAACCAGCCAAAATATTTA 121 AAGCCAGCTTACACCTGTGCAAAAAACAACAACAACAACAACAGCACTTCTTTGACCCTT >>>>> 181 TACACAGCGAGTTTCAAGAGTAATCAGATAAAGCTTGCAAAATAGAAAGAGAAGGAGGTG >>>>>>>>>>>>>>>> 241 AGAGAAGCTAGGTGGCACTTACCCTGACAGTGCTGGGCCGGGGGGGTATTTAGCCACTTG 301 GTGATGGTGTACACCTCACTGGTTTGTGGTGTACTTATGTAGTTTCCATCTCTGCTCACC 361 ACACCCTCTAACATCCCAAACAAGGAGCCTGGGCTGCACTCGGATCGGTAGCCTCACTCC 421 ACTAAAGCCTTATAGGGATGACTGGGAGAAAACACTCCAATCCGGGCAGCACCAGGGCGG 481 CCTCTTGCCTCCTTACTCTGGGCATTCCAATGGACCTTTTCCTTTTTTCTCCTCAGAAAT 541 TGTGGGGACTTCACTATGCACGAGAATAAACACAGGGCTGGGCCTGATCTGCTACACAGA **<<<<<<<<<<<<<<<<<<<<<<<< 601 CGCTCACCACGGCCCACACCTGAGAAGGAATGGCAAAGACATTTGGTCACTTAAGTACAT 661 AGGTCACACGAGTTTAAAAGTTTAAACTCCCAAGCTGTACTGTAAAAACAAAGCAAAAAG 721 TAAATAAAATAAAATCTTATG

BACK UP CNS1 Primer []

PRODUCT SIZE: 466, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00 TARGETS (start, len)*: 201,350 1 TAGGTAGAAAATGTATCTTTCAGAAGGCACGTCTCTAGGAAAATCAATCCTCACAATCAC 61 ATTTACAGAAATGTAGATCAAATCTTTCTCAGAAAACACTCTAACCAGCCAAAATATTTA 121 AAGCCAGCTTACACCTGTGCAAAAAACAACAACAACAACAACAGCACTTCTTTGACCCTT >>>>>>>> 181 TACACAGCGAGTTTCAAGAGTAATCAGATAAAGCTTGCAAAATAGAAAGAGAAGGAGGTG >>>>>>>>>>>>> 241 AGAGAAGCTAGGTGGCACTTACCCTGACAGTGCTGGGCCGGGGGGGTATTTAGCCACTTG 301 GTGATGGTGTACACCTCACTGGTTTGTGGTGTACTTATGTAGTTTCCATCTCTGCTCACC 361 ACACCCTCTAACATCCCAAACAAGGAGCCTGGGCTGCACTCGGATCGGTAGCCTCACTCC 421 ACTAAAGCCTTATAGGGATGACTGGGAGAAAACACTCCAATCCGGGCAGCACCAGGGCGG 481 CCTCTTGCCTCCTTACTCTGGGCATTCCAATGGACCTTTTCCTTTTTTCTCCTCAGAAAT 541 TGTGGGGACTTCACTATGCACGAGAATAAACACAGGGCTGGGCCTGATCTGCTACACAGA
 * || Sequence || Position || Length (bp) || Tm C || GC% || any || 3' ||
 * Left primer || TGACCCTTTACACAGCGAGTT || 173 || 21 || 59.79 || 47.62 || 2 || 0 ||
 * Right primer || CTTTGCCATTCCTTCTCAGG || 638 || 20 || 59.81 || 50 || 3 || 2 ||
 * 601 CGCTCACCACGGCCCACACCTGAGAAGGAATGGCAAAGACATTTGGTCACTTAAGTACAT <<<<<<<<<<<<<<<<<<<<**
 * 661 AGGTCACACGAGTTTAAAAGTTTAAACTCCCAAGCTGTACTGTAAAAACAAAGCAAAAAG**
 * 721 TAAATAAAATAAAATCTTATG**


 * Ran an in silica test on both primers and both matched up to the proper sequence on zebrafish genome**


 * 9/15/11**


 * Oligonucleotide primer dilution**


 * 1. microfuge oligonucleotide primer to ensure its at bottom of tube**


 * 2.diluted primers to create a 100µM stock sol of each primer**
 * -added 219µL of nuclease free water to sense primer 1**
 * - added 249µL to antisense primer 2**
 * - added 215µL to sense primer 1**
 * - added 233µL to antisense primer 2**


 * 3. made a 10µM by adding 10µL of both forward and reverse primer working solution with 80µL of nuclease free water**
 * vortexed solution and kept in freezer**


 * 4. came back to lab later and mixed up a stock solution of Standard Accuprime //Taq// Polymerase Reaction**
 * -105µL molecular grade water**
 * -12.5µL of 10x Accuprime polymerase buffer II (contains MgCl2 & dNTPs)**
 * -2.5µL of Genomic DNA in TE(~0.10µg)**
 * - 2.5µL Primer mix in working sol (10µM each)**
 * -2.5µL of Accuprimer //Taq// Polymerase**

-this continuse for 40 cycles resulting in exponetial increase of 1.1x10*12 more copies than original sample so 40 sets of intermediate template strands and 1.1x10812 sets of precise length template strands -by the 3rd cycle fragments of the precise length are generated -taqpolymerases enhance specificity used cloumn 2 A-D A=60.00 B=59.8 C=59.2 D=58.3
 * 5.Ran PCR with BIO-RAD iCycleriq**
 * PCR produces a large amount of my specific DNA from a small amount of nonspecific DNA (The controlled enzymatic amplification of themplate DNA molecule containing a specific DNA sequence of interest)**
 * -PCR makes use of compelementary DNA strand hybridization and DNA strand synthesis via DNA polymerase**
 * -before the DNA can be amplified the sequence of DNA upstream and downstream of the region of interest to make** oligonucleotide primers **that serve as starting points for DNA replication (these primers are needed becuase** DNA Polymerase **requires double stranded DNA)**
 * -the two complimentary strands are melted apart into single strands before synthesis begins so the primers proved a double stranded starting point for DNA polymerase**
 * -the DNA polymerase used in PCE must be thermally stable bc the PCR runs between 60 and 94C**
 * -1st step heats the sample to 94C to** denature **the DNA-hot start prohibits rxn of enzyme while rxn tubes are being set up**
 * -2nd thermocycler drops to 50-60C allowing primers to** anneal **to separated strands -however the two original strand may reanneal together and compete with primers**
 * -3rd the samples are heated to 72C for DNA polymerase to extend the primers and make complete copies of each template DNA strand-DNA polymerase works most effectively at this temp** Extension step**

9/20/11 Agarose Gel Exraction (to characterize the reaction specificity and confirm the amplicon length is correct)

1. Made a agarose gel with 50ml of 1xTAE solution and 1g or agarose gel concentration 2. heated until all agarose concentrate was dissolved 3. added 2.5µL of GelGreen to solution 4. poured gel and let stand for 10 mins 5. covered gel apparatus with 1xTAE solution 6. added 5µl of 100bp generuler to second loading lane 7. mixed 5ul my zebra fish DNA with 1ul of tritrack 6x loading dye lane 3 is DNA 1 (60.00) lane 4 is DNA 2 (59.8) lane 5 is DNA 3 (59.2) lane 6 is DNA 4 (58.3) 8. ran electrophoresis at 100v 9. 4 bands appeared around 500bp which is the brightest band on ladder. I believe the bad ladder definition on the left is due to the fact that it took 45mins to get my picture.

10. cut out bands at wells 3 and 4, and put them in a centrifuge tube which initially weighed 1.12g after adding the agarose and DNA it weighs 1.48g (.36g of gel) 11. cut out bands at wells 5 and 6, placed in a 1.1g centrifuge tube. After adding agarose and DNA the tube weighed 1.48g (.38g gel)

9/26/2011 Proximal Promoter PCR amplification and Agarose Gel Extraction -in presense of chaotropic salts (sodium iodide) DNA binds to glass particles

1. added 1080ul of QX1 buffer to the .36g of gel 2.allowed to incubate at 55 C for 5 minutes 3.vortexed the QIAEX II suspension for 30s 4. added 10ul of QIAEX II to gel solution 5. incubated at 50 C for 10mins to allow the agarose to solubilize and DNA to bind to silica beads-DNA binding only occurs when solution is yellow-optimal pH 6. vortexed every 2 mins 7. centrifuged for 30s and discarded supernatant 8. washed pellets with 500ul QX1 wash buffer, vortexed, centrifuged and removed supernatant which removed residual agarose 9.washed pellet twice with 500ul of wash buffer PE, resuspended by vortexing, pelleted with brief centrifugation, and removed supernatant to remove the excess salt 10. air dried the pellet for 10 minutes 11. eluded DNA by resuspended in 30ul of TE buffer and incubated for 55 C for 5 minutes 12. spun the tube and removed supernatant which now contains the DNA 13.stored at -20 C

10/02/2011 LAB 4: Direct cloning of the CNS amplicon into an entry vector using BP clonase

Characterization of the PCR product (CNS amplicon) quantity to flourometric analysis 1. calibrated flourometer 2. made Quant-IT BR working solution by diluting 3ul of dsDNA BR reagent into 600uL of dsDNA buffer 3. made calibration standards 1 and 2 by adding 10ul of each into 190ul of working solution 4. calibrated with these calibration standards 5. made sample for qualification by adding 2ul of purified PCR product to 198uL of Quant-IT BR working solution 6. let stand at room temp for 2 min 7. according to the Quibit flourometer, there are 0.0172ug/mL in my solution which shows that I have DNA from my purified agarose product

10/3/2011 Cloning of CNS amplicon (PCR product) into an entry vector using BP clonase 1. added 7.5ul of attB PCR product, .5ul of MD01 (pDONR 221 150ng/ul), and 2ul of BP clonase II enzyme/buffer mix 2. vortexed briefly, and incubated for 25 C for 1.75 hours 3. added 1uL of 2ug/uL Proteinase K solution which degrades the BP enzyme so a reverse reaction does not take place 4. incubated for 10 minutes at 37 C for 10mins 5. stored BP reaction at -20 C

10/04/2011 Transformation of BP clonase reaction product into TOP10 chemically conpeteny E. coli

1. added 1ul of BP clonase reaction to microfuge tube and placed on ice 2. Thawed TOP 10 cells and added 20ul to the BP clonase reaction 3. incubated on ice for 30mins 4. heat shock DNA into the cells for 30s at 42 C 5. put heat shocked sells back on ice for 2mins 6. aseptically over flame added 250ul of S.O.C medium to cells 7.shook cells for 37 C for an hour 8. spread 200ul of transformed cells onto prewarmed kanamycin plates 9. incubated plates overnight 10. did not see any colonies

10/06/2011 Retried transformation above and still saw no colonies

10/11/2011

Subcloning the CNS into an expression vector using LR clonase

Today I was going to retry extracting DNA from my other gel, because I had a low quantity of DNA. However, when I went to the warm room to discard my old plates, they each had a bacterial colony on them. So I will proceed to the next step.

1. added 5ml of LB broth and 1ul of kanamyacin 2. touched colonies with sterile stick and placed in conical tube with broth 3.placed in warmroom overnight, leaving cap loose

10/13/2011 Subcloning the CNS into an expression vector using LR clonase

1. added 1.7mL of E. //coli// in LB media with .3mL of glycerol to yield a 20% storage solution which is stored in the -80 freezer 2. centrifuged 1.5mL of E. //coli// overnight culture and disposed of supernatant 3. resuspended pelleted cells in 250ul of P1 buffer with RNAase A added, and vortexed until mostly no clumps of bacteria were seen 4. added 250ul of P2 buffer and mixed my inverting 6 times, the tube should have become clear but it did not...I think becuase I had alot of bacteria in the tube (P2 buffer contains a getergen sodium dodecyl sulfate or SDS and a base (NaOH). Ocassionally SDS may precipiate 5. added 350ul of N3 buffer which neutralized the solution and mixed by iverting 6 times 6. centrifuged for 9 minutes which resulted in a white pellet of cellular debris and chromosomal DNA 7. transferred the supernatant which contained the plasmid DNA to a QIAprep spin column 8. centrifuged for 45s and discarded flow through 9. added 500ul of buffer BP binding solution, centrifuged for 45s, and discarded flow through 10. added 750ul of ethanol containing wash solution buffer PE, centrifuged for 45s, discarded flow through 11. centrifuged and additional 1 minute to remove residual waste so and remaining ethanol wont inhibit the following enzymatic reactions 12. transfered the QIAprep column to a new microfuge tube and eluted with 50ul of elution buffer, centrifuged for 1 min, and stored the flow through containing plasmid DNA in -20 C freezer for further use

10/23/2011 Confirming the correct size of cloned CNS insert with restriction enzyme digest

1. determined that the CNS insert would be cut in two places by the restriction enzymes ApaI (at 567bp) and EcoRV (2998 bp) by going on [|www.tools.neb.com/NEBcutter2/index.php]

Subcloning the CNS into an expression vector using LR clonase-LR cuases attL1 and attL2 sites to recombine with attR1 and attR2 in destination vector to pick up flouresence BP causes attb1 and attb2 to recombine with attP1 and attP2 to pick up the gene of the CNS

1. added 18ul of nuclease free water, 2.5 ul of 10x NEB buffer#4, 2.5ul of 10x BSA, 1ul of plasmid DNA, and 1ul of ApaI enzyme 2. incubated at 25 C for 1 hour 3. then added EcoRV and incubated at 37C for an hour 4. ran gel (1% agarose, and used 1kb ladder) 5. did not see any bands of DNA

10/24/2011

Since I did not see any DNA in my gel, I am going to try the mini-prep again tomorrow, so first I need to regrow some bacteria

1. I added 5ml of LB broth to two conical tubes, added frozen stock of transformed ecoli, and 5ul of kanamyacin 2.left on shaker in warm room overnight

10/25/2011 Redoing miniprep from before with new cells

1. spun down ecoli cells 2. added appropriate amounts of P1,P2, N3 3.used spin coulumn and added PB, PE, and EB while centrifuginh at intervals

Confirmed from [|www.tools.neb.com] that the restriction enzymes would not cut my CNS, so there should be two bands. One at 2330 BP and one at 590BP 4. ran gel, had awesome ladder...and still no DNA showed up. 5.checked my amount of DNA on the flourometer, and it was too low to be detected. 6. I am going to grow more ecoli in the warm room over night, and then try again tomorrow.

10/27/2011 1. Redoing miniprep 2. checked with quibit flourometer to see if I have DNA, which I do 3. ran gel still nothing.

11/1/11 1. I made a 1% agarose gel 2. loaded DNA from third and second miniprep before restriction enzymes were added to see if there was any DNA 3. also added DNA from third miniprep which was supposedly cut 4. No DNA showed up again 5. I'm going to try and grow a larger amount of bacteria in attempts to extract a larger quantity of DNA. I feel that I have been getting DNA from the miniprep but it is too small of a quantity to show up in the electrophoresis 6. added alot of frozen down bacteria to 25mL of LB broth and 25ul of kanamyacin, placed in warm room

11/2/11 1. I let the bacteria grow overnight and the solution was much more cloudy that in previous attempts 2.did miniprep again 3.added restriction enzymes 4. stored DNA in freezer

11/3/11 1.I'm running a new gel to hopefully get bands of DNA 2.I expect to see some around 600 and some around 2300 3. I saw one very faint band, so we think that I may have had too much bacteria which clogged the miniprep filter 4.Kayli and I also pulled needes, 6 with filament and 6 non filament to prepare for morphilino injection

11/8/11 1. Tried miniprep again 2.cut with restriction enzymes

Morphilino 1. Dr. Balza and I got for tanks of fish. I filled them with some fish, with a ratio of 2 females to 1 male. 2.Waited for them to mate, but they did not

11/10/11 1. I ran the electrophorsis gel again with the new mini prep cut enzymes, and this time I did not see a band again

Morphilino 1. Kayli 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 3. 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...CX 36??? 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

11/21/11

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

11/30/11 Injected embryos with FOG, but we only got 3 morphants injected correctly but they later died

12/1/11 1.did mini prep 2.checked plamid content with qubit flourometer, which read around 1.6 3.however, when we ran the gel we didn't see any DNA 4.prepared injection cocktail anyways

12/2/11 1.injected mCHERRY insulin glowing fish with the MEF2C cocktail that is supposed to glow green