pcr cloning

[boa]

I have no clue where to start but I think it is safe to say that my forward primer could be TAC and my reverse primer TTA (complementary sequences of the start and stop codon) but I dont know what to do after...the problem seems very overwhelming to me! I would like someone to provide me some hints to tackle this!

Thanks everyone!Thank you for your help!

 

Question :

Write the sequence of two oligonucleotides that will allow you to clone the coding region of gene x

in the vector pQE60 using PCR . The coding sequence must be in frame with the ATG of the vector. The histidine tag must be present

at the C-terminus of your recombinant protein. The oligos must be as short as possible but must

hybridize with 20 nucleotides of the template sTrand. The start and stop codons of gene X are

underlined.

 

Coding sequence of gene X

GTCGATCAAT ATGGAACATG TTTACTCCAA ACCACCGCAC ACCAATTATG

GAAACCAAGC CGGAAAAGAA TTCCGGTGGA GAGCGAAAAA AAAGGATTCC

GAATCGTGAA CTGCCAAAAA CATTTTGAAG CCAACGATTC CGACGTCATC

CTCGCCACCC TAGCTAAATC AGGCACCACT TGGTTAAAAG CTCTTCTCTT

TGCTCTCATT CACCGACACA AGTTCCCAGT TTCTGGCAAG CATCCTCTTC

TGAAACAGCA GTAGCAGCGT TTAAAGGGAA GTTTATT

 

Oligo #1 5' ________________________________________…

Oligo #2 5' ________________________________________…

 

Nco1 = CCATGG BamH1 = GGATCC BglII = AGATCT HindIII = AAGCTT

 

pQE60 (RBS)CCATGGGAGGATCCAGATCT(blackbox) TAAGCTTCCGCATAATTAGCTGAG

Additional Details

The start and stop codons of gene X are the first ATG and the last TAA

 

underlined in vector pqe60: the first ATG

 

 

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I have no clue where to start but I think it is safe to say that my forward primer could be TAC and my reverse primer TTA (complementary sequences of the start and stop codon) but I dont know what to do after...the problem seems very overwhelming to me! I would like someone to provide me some hints to tackle this!

Thanks everyone!

Edited October 2, 2012 by boa

[CharonY]

Check out the requirements of your primer. First, it has to hybridize with 20 bp. So the minimal length is 20. You do need to add a His-Tag on the C-terminus (which primer would that be?). So far you offered 3 bp for each primer only.

In addition you may want to think about compatibility with the vector (though you can always blund-end ligate).

[boa]

Check out the requirements of your primer. First, it has to hybridize with 20 bp. So the minimal length is 20. You do need to add a His-Tag on the C-terminus (which primer would that be?). So far you offered 3 bp for each primer only.

In addition you may want to think about compatibility with the vector (though you can always blund-end ligate).

 

Ok, so I tried to make primers according to gene X...

so for the forward primer(oligo 1) I have : 5-ATGGAACATGTTTACTCCA-3

and for the reverse primer (oligo 2) : 5-TTAAACGCTGCTACTGCTGT-3

 

Am i doing this right? Also, how do I include the his tag? I dont understand what the c terminus thing is about...

Thanks!