Need HELP Willing to pay - (May/25/2011 )

Hi,

I need some help with this problem and am willing to pay some through paypal for help that I desperately need. Below is a paragraph from a paper I read. I am working for someone who is interested analyzing the LOX enzyme structure and fucntion. I need help on how to get the LOX gene into a plasmid that can be expressed in E coli.
I don't understand what is a QUICK-Clone cDNA library. Is it a copy of all the mature mRNA in a cell? How does one find the sequence of interest in the cDNA and amplify it so it can be put in a plasmid. I don't understand how to make primers. Are the complementary to flanking sequences or the sequence at the end of both ends of the gene?

Any help one could quickly offer would be awesome and I can pay you a little money for help through paypal

Generation of pLOX01 construct
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 a
double PCR process. The forward and reverse primers (Midland
Certified Reagent Company) were 50-GTTCCTGCGCTCAGTAACC-30
and 50-CACCAGGCACTGATTTATCC-30 . 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 50-CTAGGGGCTAGCGACGACCCTTACAACCCCTA-30 and 50-
TTAGGGAAGCTTATACGGTGAAATTGTGCAGCCTG-30 . The isolated
LOX gene was ligated into the pET21a vector (Novagen) multiple
cloning site to generate the pLOX01 plasmid. The site of insertion
was the NheI site at the 50 end and the HindIII site at the 30 end.
Generation of the pLOX02 construct
The LOX gene was isolated from plasmid pLOX01 via PCR using
the primers MLOXF (50-AAGGGGCATATGGACGACCCTTACAACCCC-
30) and MLOXR (50-AAGGGGCTCGAGATACGGTGAAATTGTGCAGCC-
30) (Invitrogen) and ligated into the NdeI (50) and XhoI (30) restriction
sites of pET21b to generate the pLOX02 plasmid. The plasmid
was transformed into DH5a E. coli cells and isolated using a
QIAGEN plasmid isolation kit in sufficient amounts for sequencing
and transformation into an expression cell line. The gene sequence
was verified by sequencing at SeqXcel (San Diego, CA).

Before you start anything you should really really find out about primer design and cloning... it will be very important that you do so before you come to do the experiments!

If you don't know the information it will cost you lots of time and lots of money to fix things!



A cDNA library is the mRNA from cells turned into cDNA. You should be able to design primers for your gene using the first 20ish bases of the sequence and the reverse complement of the last 20ish bases. As you are wanting to express it in bacteria, you will only want the coding sequence, which starts at an ATG and ends at a TAA or other stop codon. You should be able to find this information on the NCBI website by linking to the gene as you did in one of your other posts.

If you want to clone by PCRing the gene you will need to add restriction sites to the primers. The sites must be suitable for the multiple cloning site on the vector that you are going to use (do you know which plasmid you are going to use?). Commonly these are restriction sites for enzymes such as BamH1, EcoR1 and several other less common ones. To add these, you add bases to the 5' end of the primer only! You may also want to add a Shine-Dalgarno sequence too.

There are lots of websites around that explain exactly what you need to do.

silvepa23 on Wed May 25 19:58:48 2011 said:

quick-clone library is already prepared cdna ready for screening, not a kit to extract or prepare cdna.

mdfenko on Thu May 26 20:16:06 2011 said: