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cDNA cloning by PCR


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3 replies to this topic

#1 nousheen

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Posted 17 March 2005 - 02:43 AM

:rolleyes:
i have purified a protein whose N-terminal sequence is known. If sequence of only Nterminal is known how shuould i design the primers to get the amplifcation of the desired gene can i get a cDNA clone from a single primer .what should be the primer for other flanking region of the gene?
thanx

#2 yongyk

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Posted 17 March 2005 - 11:44 AM

dear nousheen,
Can I know from which organism that you isolated your protein?
How many base pair that you know?

I need to know those information before I can know what to do.
Thank.

Regards
yongyk

#3 nousheen

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Posted 17 March 2005 - 10:32 PM

ive isolated my proteins from outer membrane of Salmonella typhimurium.for one we have sequenced 15 aminoacids and for the other 7.

#4 yongyk

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Posted 29 March 2005 - 02:18 AM

Dear nousheen,

To get the primer for other flanking region of the gene, I think is good to use bioinformatic software and make use of swissprot protein database.

Now you know 15aa (=45bp). load your know 45bp on NCBI and BLAST. From the BLAST search, the programe will show you a lot of similar protein. You can make use the information from the data base predict your reverse primer.

Good luck

Best regards
yong




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