I have no idea what to do for this question nor where to start. Please tell and
ID: 148151 • Letter: I
Question
I have no idea what to do for this question nor where to start. Please tell and show me exactly what to do step by step following the guidelines and why I should do it so that I can know the process and how to answer similar problems to this one. Please also let me if I did anything wrong as I created these primers myself and tell me especially whether or the reverse primer is correct. Here is all the background info needed:
#9) Design a strategy to subclone the PCR fragment containing this entire open reading frame (ORF) that was cut with BamH1: ( Forward primer sequence:
5’ G/GATCC - (AUG) -(AUG)-(AUG)-(UGU/C)-(UAU/C)- (UCU/C/A/G) - G/GATCC 3’ ) and (Reverse primer sequence (Reversed version of Forward Primer) ) : 5'(CCTAG/G)--C/A/G/UCU- (C/UAU)-(C/UGU)-(GUA)-(GUA)-(GUA)--(CCTAG/G)'3 ) into the pUC18 vector (see below).
(Please note that the forward and reverse primers that I created were cut out of this ONE LETTER amino acid code sequence:
MMMCWCDQFLAAAIDAAKKAGQIIRKGFYETKHVEHKGQVDLVTETDKGCEELVFNHLKQLFPNHKFIGEETTAAFGVTELTDEPTWIVDPLDGTTNFVHGFPFVCVSIGLTIGKVPVVGVVYNPIMEELFTGVQGKGAFLNGKRIKVSAQSELLTALLVTEAGTKRDKATLDDTTNRINSLLTKVRSLRMSGSCALDLCGVACGRVDIFYELGFGGPWDIAAGIVIVKEAGGLIFDPSGKDLDITSQRIAASNASLKELFAEAMMAAMM .
This was done with BamH1 restriction endonuclease sites (G/GATCC) on each end to cut the fragments.
(pUC18 vector image):
Your answer should include:
a) Does your strategy use sticky/sticky, sticky/blunt (two different), blunt/blunt, etc. ends for ligation?
(I believe BamHI is sticky so use sticky/sticky??? When would you ever use sticky/blunt since you can only use one restriction endonuclease at a time?????)
b)whether your strategy is directional (i.e. can the insert anneal in either orientation?)
c) a drawing of all possible final recombinant DNA plasmid which must include the direction of the gene ORF.
d)Please also indicate whether a colony containing your desired recombinant DNA would be blue or white if you plated your transformation on a plate containing X-Gal.
Ndel, HgiEII Narl Aatll Eco0109 Bgll Sspl Mstl Pvul BamHI lacz Xmnl Polylinker am Scal lac Pvull Pvul Avall pUC18 (2.69 kb) 2000 Mstl Afllll Avall 100 Bgll HgiEll Figure 3-25 Fundamentals of Biochemistry, 2/e 2006 John Wiley&SonsExplanation / Answer
the first step for any cloning is to design primers with your choice of restriction sites. then do PCR of your target gene with these primers so that restriction sites are included. I hope that you have done PCR with these primers for the given gene, this way you have incorporated Bam H I site in your gene in both the direction i.e 5'-3' and 3'-5'. Now then you will digest your vector and insert with the Bam H1 and ligate the insert. But this type of cloning is nondirectional one because in this way your insert can ligate in either orientation because both the ends have Bam H1. so it would be advisable to use two different enzymes for digestion to prevent wrong orientation.
a) strategy is sticky/sticky
b)non- directional
c) white because the lac z gene gene gets disrupted