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Introduction

Shanni He Biol 100K Prof. Jeremy Lee TA: Kasturi Malaviya The Ligation of EGFP cDNA into pET41a(+) I. Introduction Over the course of the last few weeks, we conducted experiments in which our goal was to successfully ligate EGFP cDNA into pET41a(+) in order to transform our recombinant expression plasmids into clones of E. coli. After growing these cultures on varieties of agar plates, we attempted to isolate our recombinant plasmid DNA and ran multiple tests in order to confirm that the EGFP insert worked. GFP was first cloned in 1994 from the jellyfish Aqueorea victoria (Prasher et al., 1992). This useful gene glows green under specific lights allowing us to see when and where proteins appear. The type of GFP we used in our lab was EGFP, which is a humanized version of GFP (Yang et al., 1996). Today, GFP has become one of the most widely used proteins in biochemistry and has sparked man `` y new discoveries in gene expression and protein targeting. In order to clone EGFP, we utilized multiple procedures of recombinant DNA technology. This technology allows scientist to produce DNA artificially by extracting DNA from two different sources and combining them into a single molecule. The first successful production was done in 1972 at Stanford University, which opened a new horizon in drug development and medical discoveries. Human insulin, which is used to help treat diabetes, was the first registered drug to use recombinant DNA technology (Johnson, 1983). II. Methods Ligating EGFP cDNA into pET41(a)+ plasmid The goal of this experiment was to ligate EGFP into pET41(a) plasmid to create a recombinant expression plasmid. We set up 5 different ligation reactions (2 ligations & 3 controls) using stock tubes containing DNA of 25 ng/ul cut pET41a(+) DNA, 7ng/ul EGFP insert DNA and 25 ng/ul uncut pET-41a(+)/EGFP recombinant plasmid DNA. Our first ligation (ligation #1) consisted of a 1:1 molar ratio of pET-41(a) ...read more.

Middle

to make enough mix for a total of 20 ul for each PCR (71.4 ul nuclease-free H2O, 12 ul 1X PCR buffer, 9.6 ul 800 uM dNTPs, 6 ul 0.5 uM pAD1sense primer, 6 ul uM pAD1anti primer and 3 ul 2.5 units Taq polymerase). We then pipetted out 18 ul of master mix into each of our five tubes as well as 2 ul of each appropriate DNA template. We then incubated our samples in the thermocycler in the following parameters: 95°C for 10 mins 95°C for 1 min 56°C for 1 min 72°C for 1.5 min 4°C (hold, to suspend reaction/avoid degradation of DNA) We then ran our 5 DNA samples through a 40 ml 0.9% agarose gel at 60 V before turning it up to 110 V to allow our DNAS to run faster. Virual Cloning and Sequence Analysis The purpose of this lab was to use online resources to determine the nucleotide sequence of our EGFP/pET41 recombinant plasmid, the primer locations and size of the PCR produce and to design a restriction digest experiment to future prove the identity of our expected DNA sequence. To do this procedure, we used a variety of online tools. First, we used a nucleotide database to find the pEGFP-N1 plasmid nucleotide sequence. Next we used the WatCut restriction analysis tool to find the cut sites we used to cut the EGFP fragment out of the pEGFP-N1. To find the pET41(1) vector sequence, we used the LabLifeVector Database. Based on all the information and sequences we found from these databases, we then virtually “ligated” our EGFP fragment into the cloning site of the pET41(+) plasmid sequence to generate our entire recombinant plasmid sequence. From here, we searched for the location of our two primers, pAD1sense and pAD1anti, to determine the size of the PCR product. To further confirm the identity of our product, we designed a restriction enzyme digest experiment using WatCut and two different enzymes that would cut. ...read more.

Conclusion

The second band for a double non-recombinant would also be very feint and very small so there?s a possibility it wouldn?t show up clearly on the gel either. The undigested samples would run very slowly, since the DNA would be uncut and the size would be quite large. If given enough time and supplies, a second trial would have yielded much more accurate and informative results. We would then be able to identify the results of our unknown (blue) sample of whether or not it is recombinant or non-recombinant. Polymerase Chain Reaction (PCR) Based on our results, our PCR reactions were all pretty successful. The outcome of the ?blue? band also concludes how our unknown is most likely recombinant, since there is a distinct band very similar in size next to our ?green? band, which contains a recombinant plasmid. Our ?red? band yielded nothing, which is expected since it was non-recombinant, and our positive and negative controls yielded the expected results as well. A non-recombinant band wouldn?t show up very clearly since it would be very large and would travel very slowly down the gel. The fact that there were two bands in our ?blue?, ?green? and positive samples show that the ligation of EGFP was successful and that it was cut by two restriction enzymes. Virual Cloning and Sequence Analysis Through sequence analysis, it was concluded that the restriction enzymes BpmI and NciI could also be used as reagents for our PCR product. Based on the sized calculated, a single digest with either enzyme would produce two visible fragments (hypothetically) along with a third fragment of very little size that would probably not be seen on the gel. Additionally, BpmI and NciI have restriction site with a nucleotide count of only 1 difference from each other. This would mean that a double digest with these two enzymes would not display the expected 5 fragments on a gel but only three since a single base pair would not show up on the gel and the 30 bp would probably be too small to show up on the gel as well. V. ...read more.

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