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!
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
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
The start and stop codons of gene X are the first ATG and the last TAA
underlined in vector pqe60: the first ATG
your forward primer would be exactly the same sequence of gene x given... u just need to select some 20 bases upstream of your ATG sequence .. n it is your forward primer.
for your reverse primer.. you got to select the last 20bases of your geneX which would include the stop codon. then you got to add 6 codons for histidine. and then you got to create the reverse complementary sequence of that. you can do that manually.. or u may use some online softwares as well