Question

2 Addendum 1 to this lab provides information on the location of the EcoRI, and Sspl cut sites in your BlueScript vector and the 1.3 kb EcoRI fragment. Using this information, (a) draw the two possible restriction maps for the recombinant pBlueKan plasmid that you constructed (see below). There are two possible maps because the 1.3 kb fragment could have been cloned in either orientation relative to the vector, Next, (b) predict the sizes of the EcoRI. and Sspl frements that you should get when the pBlueKan recombinant plasmid is digested with each one of these enzymes (see below). (n) First, sketch the two possible recombinant pBlue kan plasmids. Use a thin line for the nortion that represents the pBlueScript cloning vector and a thick line or box to represent the cloned 1.3 kb fragment. Include all of the EcoRI, and Sspl sites and give the base pair location at which each occurs. Note that the size of the recombinant plasmid is the sum of the sizes of pBlueScript (2959 bp) and the 1.3 kb fragment (1282 bp). - Recombinant Plasmid #1: (5 points) =p Blue Seni = 1.3ub 440 Pregne 1220 Sspl psull' 4241 be - be L bp 2959 2062 Sspl be Recombinant Plasmid #2: (5 points) PSull 4241 be

Answer 1

Ssp? (2848) Ssp I (440) 2959be (701) Kan a Ssp (780) 1282 bp

i Ssp (4130) SSPP (440) ТЕся53 4241 (701) 4241 sspl (1481) EcoRI (1983) Recombinant Plasmid #1 441 БР s spl (440) to EcoRI Hisspi bp (1203) Eco RP (1983) Recombinant Plasmid #2 4241 be

The Kanamycin Gene insert has a Ssp I site, the position of which is relative to the orientation of the insert. This position is different in the two plasmids. The position of the SspI site on the pSK- vector backbone is changed as an additional 1282 bp of DNA has been inserted. The EcoRI site on the pSK- plasmid is duplicated as either end of the 1.3 kb insert is now an EcoRI site.