Nolte,+Rachel

Bio 360 Lab Notebook Rachel Nolte

__**Lab 1: Overview of transgenesis strategy, genomic DNA isolation, and bioinformatics**__ Lab partners: Tyler Kulper and Rebecca Schulz

09/01/2011

1. Zebrafish was anesthetized in 200mg/L tricaine. 2. Head and fins were removed, and zebrafish was cut into small pieces. 3. Tissue was frozen with liquid nitrogen and pulverized into powder. 4. Tissue was added to lysis buffer, and placed on rocking platform for 45 minutes. 5. Lysis buffer solution was pipeted under ethanol. 6. DNA recovered with shepherd's crook and transferred to microfuge tube.

09/08/2011

Continuing bioinformatics portion of lab. Compared zebrafish and human sequence for pax3 gene. Found five CNS regions in the pax3 gene itself but no other CNS regions close to the gene. I will refer to these as CNS1, CNS2, CNS3, CNS4, and CNS5 named left to right on the pax3 gene. All five CNS regions have comparable regions in other species' genomes as well. The CNS regions are the red areas in the graph below, compared with the human genome alone.




 * I have decided to continue with CNS5 because of its proximity to the blue exons to the right and its shorter length**. I will also gather the information for CNS4 as a backup. The primers are highlighted in yellow, and the actual CNS is in pink.

CNS4 sequence **(as backup)**

Checked primer sequences on UCSC genome browser and saw no conflicts. Left primer: Tm=59.76 Right primer: Tm=60.77 Amplicon length: 495 bp

CNS5 sequence:

Checked primer sequences on UCSC genome browser and saw no conflicts. Left primer:  Tm=60.92 Right primer: Tm=59.11 Amplicon length: 300 bp

__**Lab 2: Genomic DNA characterization & PCR**__

Lab partners: Tyler Kulper and Rebecca Schulz

09/06/2011

Prepared a gel for electrophoresis. We attempted to determine if the DNA we harvested from the zebrafish is usable. We prepared the gel with 1% agarose. The lanes were filled as follows: 1-DNA ladder 2-our DNA harvested from zebrafish (Lab 1) 3-control

Our DNA and the control failed to move through the gel. The ladder worked, however. The 1% agarose should have been 0.5% because of the large size of genomic DNA. We also may not have placed DNA in the track from Lab 1.

__**Lab 3: Proximal promoter PCR amplification & agarose gel extraction**__

09/15/2011

Dr. Balza ordered the oligonucleotide primers from Integrated DNA Technologies, Inc. The primers are freeze-dried, so the primers must be reconstituted. The oligonucleotide primers were diluted in molecular biology grade water to make a 100 microMolar stock solution of each primer. I diluted all four of the primers (left and right, 4 and 5) A working solution was prepared by adding 10microLiters of the left primer, 10microLiters of the right primer, and 80microLiters of water. I created a working solution only of the CNS5. The tubes were stored in my box in the freezer.

9/18/2011

__PCR #1__

A polymerase chain reaction was carried out using my primer working solution for CNS5, which was created on 9/15/2011.

The Standard Accuprime Taq Polymerase Reaction stock solution was prepared with the following: 100microLiters total volume of solution mixed. The total volume was divided into 4 PCR tubes for 25microLiters each. 84microLiters of water 10microLiters of 10x Accuprime polymerase buffer II 2microLiters of genomic DNA in TE buffer (used control from Lab 2 rather than the DNA harvested in Lab 1. DNA harvested in Lab 1 did not show up in agarose gel electrophoresis. 2microLiters of working solution created on 9/15/2011 2microLiters of Accuprime //Taq// polymerase

The PCR protocol was run as follows:

1. Hot start at 95.0 C for 3 minutes 2. a) 95.0 C for 30 seconds b) Gradient temperatures for 30 seconds Tube 1: Placed in row A at 61.0 C Tube 2: Placed in row D at 60.3 C Tube 3: Placed in row E at 59.8 C Tube 4: Placed in row H at 59.0 C c) 72.0 C for 1min, 30s Step 2 repeated 40x. 3. 72.0 C for 2min 4. Cooldown at 4.0 C

The cycler completed the PCR reaction. The tubes were removed from the cycler and placed in my box in the freezer.

9/26/2011

A gel was run by using 2% agarose concentration. Both the 1kb ladder and the 100bp ladder were used for comparison. My primers did not work, and the PCR product could not be seen under UV light.

__PCR #2__

A polymerase chain reaction was carried out using my primer working solutions for CNS4 and CNS5.

9/27/2011

A gel was run by using 2% agarose concentration. Both the 1kb ladder and the 100bp ladder were used for comparison. Nothing showed up on the gel except the ladders.

__PCR #3__

A PCR was started using both my primers and Becca Schulz's primers, but the cycler failed. The cycler had an error in a hot cycle and paused there for the remainder of time.

9/29/2011

__PCR #4__

A PCR was carried out using both my CNS4 and CNS5 primers, as well as Becca Schulz's primers for nkx2.5.

Set A

1-CNS4-RowC-59.4 Celsius 2-CNS4-RowD-58.7 Celsius 3-CNS4-RowE-57.6 Celsius 4-CNS4-RowF-56.9 Celsius 5-CNS5-RowC-59.4 Celsius 6-CNS5-RowD-58.7 C 7-CNS5-RowE-57.6 C 8-CNS5-RowF-56.9 C

Set B 1-nkx2.5-RowA-60.1 C 2-nkx2.5-RowB-59.9 C 3-nkx2.5-RowC-59.4 C 4-nkx2.5-RowD-58.7 C

I ran the resulting samples through 2% agarose gel electrophoresis. All of Becca's samples worked, but my samples still failed.

10/6/11

I am using Dr. Balza's primers because my attempts at PCR with my primers failed. From Lab 1, by using in silico PCR, the amplicon length and Tm is found.

E298 (referred to as Primer #2)

Forward Primer GCGTGGTTCGCATTAAGCAGT

Tm:64.6C

Reverse Primer CGCTGTGTACAATCCGAGCCG

Tm:68.2C

GCGTGGTTCGCATTAAGCAGTggaggtgccaaaaatcgctcattctgcaa accacacgaaccgaatcgactttggaagaatatatgatagcgatttacag ccacatgtagaattaaacaaatacatttatatcgaaatttaatctctaag tgtatttaaaaaaatctttaaccacaagagtaaaatgatattaacaatgc acatgaaatcacacatgcataaaagtttggtttatagtttacaaaaaagt agtctaaaacattaaagctttaaccttcctcagggaggattcacgtgaac taataaacacgccagatatggtttggtttaattaagccccagtcactgaa aggcccgggttagtgacagcctttccatgtagtaatcacgagccagtcac agttgaaaatatatgcaaattacgagtgcagtttgtggtttggcaaatag cgatcacattataaaccacgatgtaaatataccctacaactgcaaggctt tcttcattctcatttcactcaatttttattttatagacaacgagtttcct catgatatggcatacatttactttaaacggtaatgaaaaagcaattatgg tcgcaatcagttatatccaaatcctctttgattatccgttacactgtatt cagactggcgctataaaattaagaacaattcgtctgtaatgattatggac cttgtttattttgggaggtggattaaaagtggatatagcataaattatcc gctaattatacaccagtgtcgcctcaattatcctcttatcaaaaatggta gccctaaatgcagcatttagtttcaattttctccctttctgtaacaagaa ctgcgtaccttgctattattaccagggatgttatttactttgccggattt aagagtgcacaagcgccgtagataaccattttccatggtggactgaactt gggagagacagaaaggcagctggtcgctcggaaagcttttggattcctac aagatatcgaacactcggagattaaaagtattgcagcaggtaagagtaaa cgtggagcattagatcgattgtttgaaggaagaaataatcctaaatgcat gcatgttgtaatgttattttctgaatatgataaaaagctgtcatattttg tttaaaggaacatttcaaatgagttcacattcgtgaccaggagtttgatc gattatatgccgctgctgctgcatgtgtgattttttttgtcaagcaagag agatcgaaaccagatacatattctgtaatagccttgccatggtacaaaca aatcttccaattatgctttactgctggcattttaagtgcttaattgatcg agattttatggtttgctacctttaaaatggcttcccttacctgcgctgac agtttggcaattaggaaataaatatagaatggctatttcatccagggact gatctataaataatttgatacaagctgacaacatgttgcagcgggcaagt gaatctgtttcagcctatcttgaccacaggaagtgtgtgtcatttaaaac ccaaagaatttacatattgctaaaggactttgaattgtgttgcgttttag gaaatgaaaataataatatacaactacgaaattaatgcattagattttta aaactacaaatattttaataatatttttaatgtaatttcggtaataaaat gcgtatgannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnaaaaggggatctgtgtgaacatgccggcgc tcgagtgcatccatgcagccttatctgagcctttacatgcaacttgttcg attcaccttcattctgaagcgcacagtttttgccagtggcccgaacagac

ctactgacCGGCTCGGATTGTACACAGCG

FOXP1(E187)

(referred to as Primer #1)

>[|chr6:35409780+35411106] 1327bp {Forward Primer TTTGCAAAACATTTTTGACTG

Tm:56.5C

Reverse Primer GATCAGCCATTGTCACCTCCCAG

Tm:67.3C

TTTGCAAAACATTTTTGACTGcaaaaaggtaaaccatgattaaagatttt ttttttttgtttcgactgtagattatgtaaattgcacatgtttttcacaa taagcagacaaaagcaaatgatagatgttaacatgactgtgtttcccttg tctaactgttatctagtcgaacaaaaaaaacatctttattccagataaac aaggtgtcagacatcagtcaatctcgcctcaatacctcgtctagattgac ttggagccaaatcgaagcaacaaacgaatgagagcctgtgcacattactc gtgaaattttaaagaagtgctgttttattttattcattgaggaatccatt ataggctgtattcctaagaagtatgtaaatgacgtgcctttcaaggtaga gccggggcagaatcctctccatcaactaatgacaccgctcattacatgca gaacagagcccaatgaaagtgctcctgtctgtggccgccgcttccctctg taaatccctatgaaatgctaatatctcacttctaattaaaggatactaag tccaaatgtacctacgtgccagcaacataaattttattaatgtttttttt tctctctcttcgtcgctacagacgcgtctaggcttcattccattaattca tatttaataccacggctgctcaagcattcttctctcttttctttttactt tcactgcctttttattttcgatcacttgtggcccacgattacaaacagat gctcattgtgagctgaagcttcattaaggggttgtgaaactatagttttt ttttgcaggtgcacaaagcttggccacagagacgtttgcacaaacagatt catacgcacacgaggtgctaaaaagatcgaatgaagaaaaaaaaagacaa caacaacactgagtaaaccggtatggcctctccgagcctgcacacatgta cagtatttacacatgtctgcatgtaaacatgtttacaaaaaacctgcagc cattctacgaactcgaccttgacgtctgcaaacaacagcctcacaaagtt cattaagagaaggttaaaaacaatgccgttccctttcaccacaaagacaa agctggctttgtgtgttgccttttcttaaacgcaaaacgcactggaaatt gatcatagttgaattaaaaagcctatctaatggttagtatgaaatacggc ctaattagggctttggttttatagcgcctgttaacgtagttatcacttac aacattgtaatgtcaaaatcaatagggattacttatgtcctttccgcctc

cattCTGGGAGGTGACAATGGCTGATC

__PCR #5__

Using new primers #1 and #2, doing another PCR.

Start time:4:37pm End time:7:50pm Primer #1 PCR tubes 1,2,3,4

Primer #2 PCR tubes 5,6,7,8

A 58.0C tubes 1,5 B 57.6C C 56.6C tubes 2,6 D 55.2C E 53.1C tubes 3,7 F 51.7C G 50.7C tubes 4,8 H 50.0C

Gel Electrophoresis start time: 4:54pm end time:6:00pm

10/8/2011

PCR #6

Using primers #1 and #2.

Start time: 11:25am End time: 2:30pm

primer #1 tubes 1-4 primer #2 tubes 5-8

A 59.0C tubes 1,5 B 58.5C tubes 2,6 C 57.5C tubes 3,7 D 55.8C tubes 4,8

Gel Electrophoresis start time: 3:40pm end time: 4:40pm

The gel showed only primer-dimers in all of the wells.

Well - correlating pcr

10/9/2011

__PCR #7__

Using primers #1 and #2, but gradient is 50C-59C. Eight pcr tubes will be used for each primer. Start time: 1:35pm, End time: 4:50pm


 * Primer #1 || Tm (Celcius) || Gel Electrophoresis Well ||


 * |||||||||||||| A 59.0 |||||||||||||||| 2 (of gel #1) ||
 * 2 |||||||||| B 58.5 |||||||||||||||||| 3 ||
 * 3 |||||||||| C 57.5 |||||||||||||||| 4 ||
 * 4 |||||||||||| D 55.8 |||||||||||| 5 ||
 * 5 |||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">E 53.5 |||||||||||||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">6 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">6 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">F 51.9 |||||||||||||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">7 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">7 |||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">G 50.7 |||||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">8 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">8 |||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">H 50.0 |||||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">9 ||


 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Primer #2 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">9 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">A 59.0 |||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">2 (of gel #2) ||

In both gels run, well 1 is 1kb ladder. Successful PCR!!! Extracted DNA from gel and placed in freezer. (DNAextraction1)
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">10 |||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">B 58.5 |||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">5 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">11 |||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">C 57.5 |||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">6 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">12 |||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">D 55.8 |||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">7 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">13 |||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">E 53.5 |||||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">8 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">14 || <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">F 51.9 |||||||||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">9 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">15 |||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">G 50.7 |||||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">10 ||
 * <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">16 |||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">H 50.0 |||||||||| <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">11 ||




 * __Lab 4: Direct cloning of the CNS amplicon into an entry vector using BP clonase__**

10/11/2011

Part 1 - Cloning the CNS amplicon (PCR product) into an entry vector using BP clonase Incubated at room Tm for 2 hrs. (2:20pm-4:30pm)

-pDONR221 -BPclonase2enzyme/buffer mix -attB PCR product

Then added Proteinase K and incubated in warm room for 10 minutes. (37 C)

Part 2 - Transformation of BP clonase reaction product into TOP10 chemically competent e. coli

Placed 1microLiter of BP clonase rxn on ice. Thawed TOP10 cells, then tapped to mix cells. Added 20microLiters of TOP10 cells to BP clonase rxn. Incubated on ice for 30 minutes. (6:45-7:15pm) Transformed DNA into cells by heat-shocking. Moved microfuge tube with cells to 42C for 30 seconds. Replaced tube back into ice for two minutes. Added 250microLiters of S.O.C. medium to cells. Put in shaker in warm room for 1 hour. Spread transformed cells on two identical kanamycin plates and incubated in warm room until 3:30pm the next day.

10/12/2011

No bacteria colonies on either kanamycin plate from the warm room. Redoing the gel electrophoresis from Lab 3. I think that I did not purify the DNA from the agarose correctly. I still have the PCR product that was successful and will use this in the gel. Start:4:00pm End:5:00pm

Lane 1 is 1kb ladder Lane 2 is 8 from Primer #1 Extracted DNA from agarose gel and placed in freezer. (DNAextraction2)

10/21/11

Starting lab 4 again with DNA extracted from the electrophoresis completed on 10/12/11. Followed protocol, but no colonies grew.

10/25/11

Tested the PCR product from both DNA extractions with the Qubit Fluorometer to determine if either were successful. The concentration of DNA from both extractions were about half of what the protocol indicates. (10-25 fentomoles per 7.5microLiter)

DNA extraction 1 .0204micrograms per 1microLiter. So for the indicated 7.5microLiters in the protocol, 4.30fentomoles of PCR product were present.

DNA extraction 2 .0136micrograms per 1microLiter.

10/27/11

Used last of PCR product and ran through 2 wells of electrophoresis gel.

11/2/11

Extracted DNA from gel ran on 10/27/11. (DNAextraction3) Ran Qubit fluorometer analysis, but concentration of DNA was too low to be found.

Ran two BP clonase reactions: A (from DNA extraction1) B (from DNA extraction3)

I had started the BP clonase reaction for B before I ran the Qubit fluorometer analysis. I followed protocol for this sample.

Since I had DNA in the first two extractions, just in too low of a concentration, I doubled the amount of PCR product (to 15microLiters) and also doubled the MD-01, aka pDONR 221 (to 1microLiter). I added the same amount of BP clonase indicated on the protocol, however. I followed protocol the rest of the transformation. When plating the cells (on kanamycin plates), I labeled them the same way, A and B.

11/3/11

Plate A has grown colonies.

__**Lab 5: Subcloning the CNS into an expression vector using LR clonase**__

Transferred four separate colonies from the growth plate into conical tubes with LB broth and kanamycin (.5microL, 50mg/mL antibiotic concentration, 5mL of LB broth). Left caps loose and taped to make sure they stayed on. Left in warm room on the shaker. I also left the growth plate in the warm room in case the bacteria did not grow in the conical tubes.

11/4/11

Checked on the tubes and the growth plate. All of the tubes are cloudy, so bacteria has grown. The growth plate appears to have two different strains of bacteria or two different species altogether. This was not apparent yesterday. The off-white bacteria, most likely the e. coli top ten cells, were the only observed colonies yesterday. The off-white bacteria are bigger in area than the new bacteria. The new bacteria is yellow, and smaller in area. Since the growth plate has kanamycin on it, both must be resistant. I am growing more plates to determine if both have incorporated the plasmid. I am referring to the species of bacteria from now on as blue (off-white) and green (yellow), by the color of tape used on the tubes.

Four more plates are being set up, to separate the different bacteria, and to determine if both are resistant to ampicillin. Each of the species are plated on both kanamycin plates and ampicillin plates.

Since I don't know for sure which species of bacteria is in the tubes, I am making more and tossing the first conical tubes.

New tubes on shaker in warm room. Tubes 1-4 Blue species Tubes 5-8 Green species

11/6/11

New tubes are all cloudy, so all of them have grown bacteria. I am following the procedure outlined to isolate the entry clone plasmid containing the zebrafish CNS by plasmid miniprep in the lab handout with no changes. I am arbitrarily using tube 4 from the blue species and tube 6 from the green species. The plasmid from both strains are stored in clear centrifuge tubes in the freezer.

11/17/11

Confirm the size of the cloned CNS insert with a restriction enzyme digest. Apal will cut upstream (bp568 in the pDONR 221 plasmid) of the insert and EcoRV cuts downstream (bp2999 from the insert). The gel of the product of a double digestion should show a 2330bp band representing the pDONR 221 plasmid and a band approximately equal to the size of the PCR product (1327bp).

Using both blue and green species. Setting up ten digests to include controls. 1) Lab handout ratios (18microL H2O, 2.5 10x NEB buffer #4, 2.5 10x BSA, 1.0 plasmid DNA, 1.0 Apal enzyme) 2) Becca's ratios (14microL H2O, 2.5 10x NEB buffer #4, 2.5 10x BSA, 5.0 plasmid DNA, 1.0 Apal enzyme) 3) single ecoRV 4) single apal 5) plasmid alone

EcoRV added after incubating at 25 C for 1hr. Tm raised to 37 C and incubated another hr. 1.5% agarose gel electrophoresis, 110 volts. No bands were detected except for the DNA ladder.

11/19/11

Redoing the restriction enzyme digest and gel. Still no bands detected.

11/20/11

Starting over by taking new bacterial colonies from freezer from the saved plates containing blue and green species. Only used one conical tube for each species, and placed back on shaker in warm room.

11/22/11

Isolated the plasmid again with the miniprep kit, and this time used the Qubit fluorometer to determine concentration. There is not enough plasmid for a digest or an LR reaction. Because I am leaving for break tomorrow, I can't start anything. Green .0374ng/mL, 1.55 fmol/mL Blue .0126ng/mL

11/29/11

Redoing bacterial colonies, and leaving for 2-3 days. Hopefully there will be a high enough concentration of plasmid after this amount of time to do the LR reaction, but there will not be enough time for much else.


 * __Lab 7: Inhibition of mRNA translation by morpholino injection__**

11/30/11

Lab Partner: Selciya Lamech The FLI1-GFP transgenic zebrafish were taken from the fishtank room. They did not spawn, so we could not continue. There was another group working on the transgene injection at the same time, and those zebrafish did not spawn either.