Lamech,+Selciya

9/1/11 Selciya Lamech lab1 :Isolation of Zebrafish genomic DNA lab partners: Emily Bolda and Angie Blasezyk. First we anesthetized one zebrafish in 200mg/L tricaine. Then we scarificed the fish using a scapel by chopping off the head and second by cutting the fish transversely into small pieces. Now we froze the tissue in liquid nitrogen and pulverized to a power with mortar and pestle. once we allowed the nitrogen to evaporate, little by little we added about 1g of the powered tissue to 7.5ml lysis buffer in a 50ml polypropylene conical tube. Then we incubated the solution for 45-65 min at room temperature on a rocking platform.

9/6/11 Lab 2 :Genomic DNA characterization & PCR Lab Partner: Angie Blasezyk. -Electrophoretic characterization allows for estimation of the average length of genomic DNA relative to know standards. In this part of the lab we prepared a 0.5% agarose gell by heating .25g agarose in 50 ml 1xTAE in the microwave for 1min until completely dissolved. Then we added GelStar stain to the concentration 2.5 mircoliters per 50ml gel. We then poured the dissolved agarose into the gel mold and place comb in one end. Once the gel has solidified, we removed the comb and covered the gell with 1xTAE buffer in the electrophoresis rig. Now we loaded about 5 microliters of the high range DNA ladder into the first lane of the gel. then we combined our DNA with 1 micro liter of 6xtritrack loding dye and loaded that into the next lane. We also loaded a DNA to compare the differences between the base pairs. We set the Eletrophorese the gel at 100v until the bromophenol blue is near the end of the gel which takes about 30 to 60 mins. After this is done we imaged the gel using the Kodak Image station 4000MM. Under the Black light our DNA was not visible, but we were able to see the DNA given to us by Dr.Balza which was degrading.

The VISTA browser provides a powerful tool for comparing genomic sequences from different species. The exons will be shown in blue, while the conserved non-coding sequences (CNS) will be highlighted red. these sequences reprensent putative enchancer elements which may contribute to the regulation of your gene expression.

Primer design increase the efficiency of amplification and subsequent cloning. 1. shorter amplicons are more efficent but larger amplicons are more likely to contain useful regulatory regions. 2. limit secondary structure for optimal amplication efficiency, limit GC content to 50-60% and limit GC stretches to <4bp, 3. ideally the primer Tm should be about 60-65 degree celius. after the addition of attB sequences will be much higher. In silico PCR is a great way to insure the accuracy anf specificity of your primer pairs before ordering them. DNA sequence for CNS 1 for PITX2A

chr14:74,472,984-74,473,258 [|http://pipeline.lbl.gov/tbrowser/tbrowser/?pos=chr14:74450789-74612887&base=danRer4&run=6&genes=refseq#&base=3&run=6&pos=chr14:74472984-74473258&genes=refseq&indx=0&cutoff=50]

>danRer4_dna range=chr14:74472984-74473258 5'pad=0 3'pad=0 strand=+ repeatMasking=noneCCTCTCTGCTGCTGCACTCCTTTTTCCCAGCCTTGTTTACTGGCGATGGGTCTGGCCGCTGATTGTCATCC[CTGCGGTAATGGCAATGCGTGCACCAGATCAATAACCATCTGCACCAATCCTCAAGCAATAACATTCAATAGGGAAAATCCATTTACTTTTACTTACCTTTCATTTCTTTCATTTTACCATAAGGACAGCCAGCC]TGGGCAGTGGGCTTATCTAGCAGGGCATTTAGCATGGGACACCAAACACCACAAGCACCTTAAGCCTGT

Primers for CNS1 PRODUCT SIZE: 231, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 3.00 TARGETS (start, len)*: 72,135 1 CCTCTCTGCTGCTGCACTCCTTTTTCCCAGCCTTGTTTACTGGCGATGGGTCTGGCCGCT >>>>>>>>>>>>>>>>>>>> 61 GATTGTCATCCCTGCGGTAATGGCAATGCGTGCACCAGATCAATAACCATCTGCACCAAT * 121 CCTCAAGCAATAACATTCAATAGGGAAAATCCATTTACTTTTACTTACCTTTCATTTCTT 181 TCATTTTACCATAAGGACAGCCAGCCTGGGCAGTGGGCTTATCTAGCAGGGCATTTAGCA **<<<<<<**
 * || Sequence || Positon || Length(bp) || Tm Celius || GC% ||
 * Sense || TTCCCAGCCTTGTTTACTGG || +24 || 20 || 60.10 || 50% ||
 * Antisense || TGTTTGGTGTCCCATGCTAA || -254 || 20 || 59.96 || 45% ||
 * 241 TGGGACACCAAACACCACAAGCACCTTAAGCCTGT <<<<<<<<<<<<<<**
 * KEYS (in order of precedence):** target >>>>>> left primer <<<<<< right primer

[|http://pipeline.lbl.gov/tbrowser/tbrowser/?pos=chr14:74450789-74612887&base=danRer4&run=6&genes=refseq#&base=3&run=6&pos=chr14:74473055-74473189&genes=refseq&indx=0&cutoff=50] chr14:74,473,055-74,473,189 CTGCGGTAATGGCAATGCGTGCACCAGATCAATAACCATCTGCACCAATCCTCAAGCAATAACATTCAATAGGGAAAATCCATTTACTTTTACTTACCTTTCATTTCTTTCATTTTACCATAAGGACAG CCAGCC []

Lab 3: Proximal Promoter PCR amplification & agarose gel extraction. PCR-gene cloning, gene mapping, DNA sequencing. Objective is to produce a relatively large amoount of a specfic piece of DNA from a small amount of nonspecifi DNA. only one template strand is needed to generate millions of new DNA molecules. 1. complementary DNA strand hydridization and DNA strand synthesis via DNA polymerase. Oligonucleotide primers that will serve as starting point for DNA replication. primers are needed cause DNA polymerase requires double stranded DNA to intiate replication of DNA or to make copies. PCR temps btw 60-95. first step in of the PCR temperature cycling procedure involves heating the sample to 94degree. at this point the denaturing occurs. then the temperature drops to 50-60 to allow the primeers to anneal to the seperated strands, then finally the temp goes to 72c for DNA polymerase to extend the primers and make complete copies of each template DNA strand.- extension step. First we briefly spin the microfuge tube containing your oligonucleotide primer to ensure that it is all at the bottom of your tube. Then we diluted our oligonucleotide primers in molecular biology grade water to create a 100 microM stock solution of eah primer. For the amount of water needed we multiplied the number of the primer listed on our tube by 10. The we vortex for a brief time. The next thing we did was create a 10mircoliter primer working solution by adding 10mircoliter of the sense forward primer and 10mircoliter of the antisense reverse primer to 80microliter of the molecular biology grade water. Vortex. After we made the primer working solution, the next step was PCR (Bio-rad thermocycler). I used the standard Accuprime taq Polymerase Reaction. So I prepared the 25microliter PCR reaction in each of four thin-wall .2 ml PCR tubes. In column A it was 60 degree Celsius, B=59.8 degree,C=59.2,D=58.3. After the PCR reaction, I had to make the Agarose gel purification of PCR amplicons. First I prepared an agarose gel of concentration appropriate for our amplicon by heating agarose in 50ml 1xTAE in the microwave until completely dissolved. I used 1.5% Agarose. Then I added GelGreen or Gelstar DNA stain to .5x concentration. Then pour gel and place comb in one end. Allowed to solidify. Remove comb and submerge it in 1xTAE buffer. Use a minimal amount of buffer to cover the gel. Then I loaded 5mircoliter of the geneRuler DNA 1kb DNA ladder in the first lane. Then combined the 5 microliter of my DNA solution with one microliter of 6xTriTrack loading dye and loaded onto the second lane of the gel. Then ran the electrophorese at 100v. after that I took a image of the result of the lanes in the gel under a UV light.

DNA extraction from agarose. in the presence of chaotropic salts DNA binds to glass praticles. can be used to purfily DNA fragment from any type of agarose, concentrate DNA, remove protiens, remove primer dimers, remover gelstar or gel greeen. inhibited by TBE buffer and be sure to use TAE bufffer

Lab4- Direct cloning of the CNS amplicon into an entry vector using BP clonase.

After the DNA was extracted from the gel, 7.5 µL of the attP PCR product, .5 µL of pDONR 221, and 2 µL of BP Clonase II were added to a microcentrifuge tube and incubated at 25ºC for 1.5 hours. Following the incubation, 1 µL of the BP clonase reaction product was combined with 20 µL of thawed TOP10™ //E.coli// and incubated for 30 minutes on ice//.// The mixture was then heat-shocked for 30 seconds at 42ºC and then cooled back down on ice for 2 minutes. The cells were then shaken for 1 hour at 225 rpm at 37ºC to recover. The recovered cells were then plated by spreading 200 µL on prewarmed kanamycin plates. The plates were inverted and incubated at 37ºC for 24 hours. Colonies were then picked from the plates and grown up in a 15 mL conical tube for an additional 24 hours in 5mL LB broth and 50µL of kanamycin sulfate diluted from 100x stock. Once the conical tube was cloudy, indicating sufficient bacteria growth, it was stored at 4ºC. A portion of the bacteria containing the product of the BP reaction was then cryogenically preserved combining 1.7mL of E. coli in LB media with .3 mL of 80% glycerol and stored at -80ºC. -ccdb kills the top 10 cells so the bacteria that survies are the ones that has the kana and not ccdb. lab 5- Subcloning the CNS into an expression vector using LR clonase.

 Using QIAprep Spin Miniprep Kit by Qiagen, the //E coli// colonies containing the entry clone plasmid with the promoter insert was lysed and their plasmids were purified for further study. The correct size of the CNS insert was confirmed via a restriction enzyme digest using Apa1 (cuts upstream of insert at bp568) and EcoRV (cuts downstream of the insert at bp2999). After the verification of the correct CNS inside the plasmid, an LR reaction was performed. In this process the CNS was sub cloned with a reporter gene that produced green fluorescent protein. The LR reaction was again, heat-shocked into TOP 10 E.Coli cells. The mixture was then heat-shocked for 30 seconds at 42ºC and then cooled back down on ice for 2 minutes. The cells were then shaken for 1 hour at 225 rpm at 37ºC to recover. The recovered cells were then plated by spreading 200 µL on prewarmed ampicillin plates. The plates were inverted and incubated at 37ºC overnight. The colonies were then picked from the plates and grown up in a 15 mL conical tube for an additional 24 hours in 5mL LB broth and 5µL of ampicillin diluted from 1000x stock. Once the conical tube was cloudy, representing sufficient bacteria growth, it was stored at 4ºC. Again, a portion of the bacteria containing the product of the BP reaction was then cryogenically preserved combining 1.7mL of E. coli in LB media with .3 mL of 80% glycerol and stored at -80ºC.

Buffer P1- this solution should have RNAase A added. SDS-alkaline Lysis soultion Buffer P2- contains a detergent and a base (NaOH). SDS may preciapte occasionally. Neutralization solution-buffer N3 Binding solution-buffer PB Enthanol- containing wash solution Buffer PE elution buffer-buffer EB