4) If you cut your genomic segment with Sau3A: 2pts HOW and WHERE (which b.p.) could it be inserted into the pGEM – 3Z plasmid? 6) Design a set of primers (12 b.p. sequence) to use in a PCR reaction that will both 4pts a- amplify gene 2 (both strands) but not gene 1, and b- add RE sites for EcoR I on both sides of gene 2. BE SURE TO INCLUDE ORIENTATION OF THE PRIMER STRANDS Starting with your plasmid with insert that you made in step 4 7) Draw the basic gel pattern of bands you would expect to see from cutting with: 6pts A) HindIII alone, B) EcoRI alone, C) EcoRI+HindII together. Label the expected size (bp) of all bands. 8) Identify all the band(s) from question 7 which you would expect to bind to a probe for gene 2 in southern blot. 2pts PGEM-3Z plasmid is commercialy available and commonly uverai Restriction t also contains 2 inserts. It has a Multiple Cloning Region (MCR) containing s where they cut. antibiotic which are shown on the plasmid map below with the base pa resistance to the a the entire E commonly used. It is 2743 base pairs without cut. It also contains 2 R) contai ning several Restriction Endonuclease sites selectable markers. (AMP and lacZ) AMP is a gene c Ampicillin. lacZ is a gene encoding a protein that produces a biu host colony blue ONLY IF THE lacZ GENE IS FUNCTIONAL ng a protein that produces a blue color and turns the entire E. col 3. PGEM -3Z Vector Madsple Cloning Region and Cleele Majp Figure 1. pGEM SZ Vector promoter and multlple doning region sequence. The sequence shown cotresponds to RNA synthesized by T7 RNA polymerase complemntary to KNA synthesined by SPS RNA polymerase and is iorthe Notㄷ The pGEMa3Z and pGE1R4Z Vedes are idential e orientation of the SP6 and T7 promoters Soal 1818 竺-) Figure 2 PGEM SZ Vector cincle map and sequence nefesence points multiple doning region SP6 RNA polymerase promoter (-17 to +3) SP6 RNA polymerase transcription initiation site ac operon sequences binding site af puc/13 Reverse Sequencing Primer 12 start codon incZ operator p-lactamase (Amp) coding region binding site of 7 RNA polymerase promoter (17 to +3 5-61 67-86 94-523; 2564-2724 104-125 108 128-144 1265-125 2677-2700 272-3 Lab 3 – Experimental systems /virtual molecular biology example RE sites. and cut patterns: all top strands are 5′ to 3′ from left to right Bam HI G GATCC G AATTC CTTAA G GGC(C C CGG GTT AAC CAA TTG Eco Rl CCTAG G GG cC Hae III Hha I Hind III A AGCTT TTCGA A CTGCA | G G ACGTC Hpa I Pst I Sau 3AGATO CTAG Use the following genomic sequence, and plasmid information, and the restriction enzyme sitesi above to answer the assigned questions. rom TGGATCCGAGAATTCTAAA gene 1 (600 base pairs) ATGAATTCGGCC Direculy linked with no gap TAAGCTTCTGCAGC ene 2 (1200 base CAACTGCAGAAGCTTGGATCC-3 pairs) This is a single strand example. You should be able to write the complimentary strand. Note orientaticn (5′ to 3′). Assume there are no RE sites in the genes. The curved line simply connects the top line to the lower line, there are no base pairs in the curved line (i.e. it reads GGCCTAAG from end of top to start of lower).