pcr

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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lara
Coral
Coral
Posts: 131
Joined: Sun Sep 25, 2005 5:10 am

pcr

Post by lara » Tue Jun 10, 2008 11:44 am

Now this has been puzzling me for sometime.The following is from the
paper titled "Quorum-sensing system influences root colonization and
biological control
ability in Pseudomonas fluorescens 2P24"
(Hai-Lei Wei,and Li-Qun Zhang).can anyone help me out with this? the
trouble is can't visualize it and without it can't proceed.....

To create a pcoI gene deletion allele, two fragments
flanking pcoI gene were amplified by PCR. One was created by primers
I473
(5'-TCGTTCGCTGCAGGAAACC-3') which included the Pst1 site and I2056 (5'-
ATGGATCCCGGAGTTTGTGCATGCCC-3')which introduced the BamH1 site and the
other was created by primers I2328 (5'-
AAGGATCCGGGTTCGGCTTCTCTGACAC-3') which introduced the BamH1 site and
I3966 (5'-ATGAATTCAATGGCCCACTCAACGCA-
3') which introduced the EcoR1 site.The standard PCR was carried
out.After being digested with relevant restriction enzymes, the two
fragments were inserted into pBSNot6.

Cat
King Cobra
King Cobra
Posts: 635
Joined: Thu Feb 14, 2008 7:40 pm

Re: pcr

Post by Cat » Tue Jun 10, 2008 10:14 pm

Normal pcoI gene:

-- Flanking region 1 -- pcoI gene -- Flanking region 2 ---

Deletion of pcoI gene:

-(Pst1)- Flanking region 1 -- (BamH1) -- Flanking region 2 -(EcoR1)--

Homologous recombination substitutes normal allele with the deletion.

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