Wagner,+Miranda

September 1, 2011

Lab 1: Overview of transgenesis strategy, genomic DNA isolation, and bioinformatics, with Nick Goldner.

First, we anesthetized a zebrafish, using 200mg/L tricaine. We decapitated him using a scalpel, and cut him into many pieces. Liquid N was used to snap freeze his body, and the body was ground to a powder via mortar and pestle. The powdered zebrafish was carefully added to 7.5 ml of lysis buffer, and placed on a rocker for approximately 45 mins at RT. The solution was removed and added to 18mL of 190 proof ETOH. There was very little DNA available, and mixed with larger pieces. We centrifuged the tube, but still had very little DNA to recover.

September 6, 2011

A 0.5% agarose gel with GelStar was prepared. 5 ul were added to each lane. Lane 1 contained a high range ladder. Lane 2 contained our gDNA. The gel did not resolve.

September 8, 2011

Today, Nick and I found primers in our gene nkx 2-5. The first CNS gene sequence:

We broke the gene up into 3 sections, each of us taking one section, and then working on the last one together.

1: Shared CNS sequence



OLIGO start len tm gc% any 3' seq LEFT PRIMER 236 20 60.05 55.00 2.00 1.00 TCATCCTCCACCTTCCAGTC RIGHT PRIMER 3539 20 59.94 60.00 8.00 0.00 CAGAGAGCTCCCTCATCCAC

2: Miranda's CNS

OLIGO start len tm gc% any 3' seq LEFT PRIMER 198 20 59.99 55.00 7.00 2.00 CCAGCCTGGTGTTCTACCAT RIGHT PRIMER 1219 21 59.02 38.10 6.00 2.00 TCGGCTTGAATAAAAGCAGTT

3. Nick's CNS

OLIGO start len tm gc% any 3' seq LEFT PRIMER 370 20 59.99 55.00 6.00 0.00 GCGGTAGTTACCTGGTGGAA RIGHT PRIMER 1462 20 59.41 50.00 4.00 2.00 CGGGTGTAGATGGACAACAA

September 15, 2011

Our Primers came in today, and since I didn't feel very good, Nick and I only diluted the primers.

We diluted the primers to make a 100 mM solution, using molecular biology grade water (nmol x 10 of molecular grade water), and vortexed briefly. Next we took 10 uL of the forward primer, and 10 uL of the reverse primer, and added it to 80 uL of molecular grade biology water. We vortexed again. This was done for each set of primers.

As we worked with the first DNA sequence (listed above), the forward DNA primer appeared to have fallen out of the tube and onto the work bench. When diluting this set, we used 20 uL of the forward primer, 10 uL of the reverse primer, and 70 uL of molecular grade water.

Our final products, three primer working solutions, were stored in the -20 freezer.

September 16, 2011

Today I ran my PCR with Emily Bolda and David Bartels. Lydia Krause taught us how to us the polymerase chain reaction (PCR).

First, I prepared my PCR reactions for each of four thin-wall 0.2 mL PCR tubes. I prepared a master mix (below), and put 25 uL per tube. -105 uL of molecular biology grade water -12.5 uL of 10x Accuprime polymerase buffer II (containing MgCl2 and dNTPs) -25 uL gDNA in TE buffer -2.5 uL Primer mix working solution -2.5 uL Accuprime Taq Polymerase

We ran our PCR reactions, using the following temperatures: -B - 62.3 C -C - 60.9 C -D - 58.7 C -E - 55.6 C

While the PCR was running, I made a gel for later. To make a 1.5 % agarose gel, I combined 50 mL of 1x TAE buffer with 0.75 g of agarose and placing in the microwave for 30-45 seconds until dissolved. Next, I added 2.5 uL of GelGreen, and poured the gel and added the comb. Solidified. Placed the gel in the cold room.

After the reaction was complete, we stored our products -20 C.

September 26, 2011

I came in today to run a gel of my PCR products. I submerged the gel in 1x TAE buffer. I combined 1 uL of TriTrack and 5 uL of each DNA solution per lane. Loaded 5 uL: -Lane 1 - GeneRuler DNA 1 Kb DNA ladder -Lane 2 - DNA @ 62.3 C -Lane 3 - DNA @ 60.9 C -Lane 4 - DNA @ 58.7 C -Lane 5 - DNA @ 55.6 C

Gel ran at 100 volts for app. 2 hours.

Next, I took my gel to the Kodak IS4000MM image station for imaging.

My gel: Gel Electrophoresis of PCR products. -Lane 1 - GeneRuler DNA 1 Kb DNA ladder -Lane 2 - DNA @ 62.3 C -Lane 3 - DNA @ 60.9 C -Lane 4 - DNA @ 58.7 C -Lane 5 - DNA @ 55.6 C

Success at 55.6 C (Lane 4).

I excised the DNA fragments from Lanes 4 and 5 and placed them in microcentrifuge tubes, then stored in the -20 C freezer.

September 30, 2011

Today, I took the gel fragments out of the freezer and weighed the tube and the tube+the gel. Lane 4 = 1.1198g - 1.1052 g = .0146g Lane 5 = 1.1534g - 1.1234g = .0300g

Next I added 3x agarose volume (L4=43.8uL; L5=90uL) of QX1 DNA binding solution to the gel, and incubated for 5' at 55 C. This dissolved the agarose. Then I vortexed Qiasex II silica gel for 30 sec to resuspend. Added 10 uL Qiasex II silica gel to the gel slice tube. The solution was incubated for 10' at 50 C to solubilized the agarose and bind the DNA to the silica. Every 2', I vortexed to keep the Qaisex II suspended. The mixture was yellow.

Centrifuged for 30 sec and carefully discarded the supernatant, using a pipet. The pellet was washed with 500 uL of QX1 wash buffer. Pellet was resuspended by vortexing, then centrifuged again, and supernatant removed.

Washed pellet with 500 uL PE wash buffer (x2). Resuspended, centrifuged, and removed the supernatant.

Air dried the pellet for 10'-15' until it was white.

Lastly, I eluted the DNA into TE Buffer (@ pH 8.0) by re-suspending in 30 uL of sterile TE buffer, incubated for 5' @ 55 C. Then centrifuged, removed the supernatant, and placed the SUPERNATANT into a new conical tube, while carefully avoiding the pellet. I stored the DNA in the -20 C freezer.

I came back after lunch for dIrect cloning of the CNS amplicon into an entry vector using BP clonase. I combined: -7.5 uL of the attB PCR product -0.5 uL pDONR221 (MD 01) -2 uL BP Clonase II enzyme/buffer mix at room temperature, and let incubate from 2 p.m.- 4 p.m. @ 25 C.

Then I added 1 uL of 2 ug/mL Proteinase K solution to the conical tube, and I let it incubate at 37 C for 10'. Next, I transformed 1 uL of the BP clonase reaction into competent E. coli.

1 uL of the BP clonase reaction was added to a new conical tube, and placed on ice to chill. A vial of TOP10 E. coli cells were thawed on ice; Kayli and I shared a vial. I added 20 uL of cells to the conical tube. The vial of cells + the BP reaction product was incubated on ice for 30 minutes.

Next, I transformed the DNA into the cells, by heat-shocking the cells for 30 seconds at 42 C without shaking. The heat-shocked cells were placed back on ice for 2'. 250 uL of S.O.C. was added to the cells, aseptically. For 1 hour, the cells were placed on a shaker at 225 rpm (37 C). Kanamycin plates were placed in the warm room as well.

I spread 100-200 uL of the transformed cells on the pre-warmed kanamycin plate, and incubated the inverted plate overnight at 37 C.

October 1, 2011

I came in this morning and took 4 colonies from my plate and placed each colony in 5 mL of LB Broth with 5uL of kanamycin. These were incubated at 37 C overnight. I wrapped the plate with parafilm and placed the plate in the cold room (4 C) until further notice.

October 2, 2011

I came in today and looked at my cultures. Nothing grew. I took 6 more colonies to grow in LB Broth and then re-ran the BP reaction as before.

October 3, 2011

This time, nothing grew on my plates. I took two more colonies from my first plate and tried to grow them in broth again. Nothing grew. Time to join forces with Nick Goldner.

November 3, 2011

We ran a BP reaction on Nick's DNA, and did the transformation. No colonies formed.

November 21, 2011

We ran a second BP reaction, and got one colony. We took a sample to grow in broth, and re-plated the bacteria.

November 30, 2011

We got bacteria to grow in broth, and also took more samples to grow in broth. Froze down cells-80 freezer. Mini-prep of the cells. Ran gel No DNA.

We added 1.7mL of E. // coli // in LB broth with 300 uL of glycerol to yield a 20% storage solution which is stored in the -80 freezer. Miniprep- We centrifuged 1.5mL of E. // coli // overnight culture in LB Broth, and disposed of supernatant. Then we resuspended the pelleted cells in 250ul of P1 buffer with RNAase A added, and vortexed until the pellet and clumps disappeared. Next we added 250ul of P2 buffer and mixed by inversion (6 times). We then added 350ul of N3 buffer which neutralized the solution. Mixed via inversion 6 times again. Centrifuged for 9'. This produced a white pellet of cellular debris and chromosomal DNA. We transferred the supernatant (containing the plasmid DNA) to a QIAprep spin column. Then we centrifuged for 45 seconds and discarded the flow through.Next we added 500ul of buffer BP binding solution, centrifuged again and discarded flow through. 750ul of ethanol containing wash solution buffer PE was added and centrifuged for 45 seconds, discarded flow through. We centrifuged for one more minute to remove any other waste. Also, any remaining ethanol is removed so it won't inhibit enzymatic reactions.We transferred the QIA prep column to a new microcentrifuge tube and eluted with 50ul of elution buffer. We centrifuged for 1 min, and stored the flow through in the -20 C freezer-this contains the plasmid DNA-hopefully. :)

December 2, 2011

Repeated the previous experiments, and ran another gel. Again no DNA.