840:153g:Projects/project1/2008/12/02

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Rush to the finish

Melissa and Tiffany analyzed the sequence that was returned from ISU for the DNA PCR product. The complete PCR product was around 2000 base pairs and the sequencing is only accurate for about 1000 base pairs. The forward sequence was analyzed in with the NCBI BLAST program and had a 100% match for the first 649 bases of the target gene. The reverse sequence was analyzed in the same way and had a 92% match with the last 241 base pairs of the target gene. There were no gaps in the analyzed parts, so there is no intron in the provided sequence. If there was more time in the semester, primers would be designed at the ends of the known front and back sequence to continue the sequencing in the missing middle portion.

Angie and Diwash isolated the plasmid pSB1A2 having the insert cytochrome p450 gene from the culture of transformed cells. We use the Gene Jet plasmid miniprep kit for the plasmid isolation. There were altogether 30 cultures, but we isolated the plasmids from 23 culture only because there were no enough space in the centrifuge. Also, it saves our time for plasmid isolation because we don't have to repeat the centrifugation process. While doing the pSB1A2 isolation, we didn't see any interface between supernatant and the pellet after the step 4 of Gene Jet protocol. We just took out 200 microliter supernatant and continue from the step 5. This may be due to the old culture because the culture was done 1 week ago. Anyway, we isolated the plasmid and we are going to run the plasmid in agarose gel in the next lab after the restriction digest so that we can varify if the plasmid has insert or not.

Sushma,Rajiv and Binu digested the cDNA with Xba1 and Pst1.Then we purified the digested sample using Qiagen PCR Purification kit.We ligated the cDNA with 3 various concentrations of vector and gene.This was then refrigerated for transformation.A part of the elute was then electrophoresed to observe the outcome of the digested sample.On the we got 3 bands.One is the Undigested band that is around 2000-2500bp.The other two are around 1200 and 1000 bp.These bands resemble the bands that we got for the digested DNA sample that we did on 11/14/2008.This might be due to misplace of the sample by someone that is why we got the same bands of DNA sample.