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cDNA for plasmid transformation PLEASE HElP


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#1 silvepa23

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Posted 24 May 2011 - 05:43 PM

Hi

Any help would greatly be appreciated. I want to study the human lysyl oxidase (LOX) protein first I need express it in E.coli. In a paper I read The gene for human aorta lysyl oxidase was isolated from a QUICK-Clone cDNA library (BD Biosciences Clontech). Is the QUICK-Clone cDNA library, the cDNA of all the mRNA produced in humans? If so how do I determine the cDNA sequence of my gene and what primers I use? When choosing primers do you look at the the first and last nucleotide or does the primer match the nucleotides surrounding both the 5' and 3' ends of the cDNA? I went to the NCBI website and choose GENE from the drop down menu. I enter lysyl oxidase and lysyl oxidase (homo sapiens) showed up. Underneath it there was a link to order cDNA clone. Is this clone, the cDNA sequence that I am looking for? link:[http://www.ncbi.nlm....=lysyl oxidase] Is the cDNA clone the plasmid that I can transform directly into the E coli?

Any help or clearifications would greatly be appreciated.

#2 bob1

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Posted 24 May 2011 - 06:15 PM

The link from that gene name should be the DNA you are looking for. If you click on the link for ordering the clone, it takes you to a page which give you some more information about the clones, including the plasmid name. You will need to check out whether these plasmids are suitable for expression in E. coli. I know that the pCR4 plasmids are used for eukaryotic expression, but I don't know about the other plasmid listed.

Your question about primers depends on what you want the primers for: cloning? PCR? qPCR?...

#3 silvepa23

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Posted 24 May 2011 - 07:02 PM

The link from that gene name should be the DNA you are looking for. If you click on the link for ordering the clone, it takes you to a page which give you some more information about the clones, including the plasmid name. You will need to check out whether these plasmids are suitable for expression in E. coli. I know that the pCR4 plasmids are used for eukaryotic expression, but I don't know about the other plasmid listed.

Your question about primers depends on what you want the primers for: cloning? PCR? qPCR?...



Thanks so much for your response.

In the paper I read it said The gene for human aorta lysyl oxidase was isolated from a QUICK-Clone cDNA library (BD Biosciences Clontech) and screened using primers selected by a computer based primer-selecting program (Primer3) and PCR. Since primers initially chosen to give socalled sticky ends for ligation did not yield PCR product, we used adouble PCR process. The forward and reverse primers (Midland Certified Reagent Company) were 5'-GTTCCTGCGCTCAGTAACC-3' and 5'-CACCAGGCACTGATTTATCC-3' . PCR was run again using the previously obtained PCR product to introduce NheI and HindIII restriction sites for vector ligation using forward and reverse primers 5'-CTAGGGGCTAGCGACGACCCTTACAACCCCTA-3' and 5'-TTAGGGAAGCTTATACGGTGAAATTGTGCAGCCTG-3' . The isolated
LOX gene was ligated into the pET21a vector

Could you possibly explain how someone screens for a gene using certain primers? And maybe give an explanation of what is going on?

#4 bob1

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Posted 25 May 2011 - 05:17 PM

It looks like they designed two sets of primers - first off they designed primers with restriction sites with "sticky ends" (overhangs when cut with a RE) on them and tried to amplify the gene. This didn't work, so they generated normal primers and used those to amplify the gene probably using primers that bind to the UTRs of the cDNA. They then used this PCR product as template to do another PCR using the primers with the restriction sites on them, so that they could cut the product and clone it into the vector in a directional manner. Often this process of generating a PCR product and then using the result as a template for another PCR is called Nested PCR.




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