pUC 18 3 kb

1. Rational behind the Question: pUC 18 3 kb BamH I EcoR I Kpn I Sac I Pst I Not drawn to scale Sma I Cut Insertion of: RF (450 bp) AF (1,350 bp) RB (1,400 bp)

2. A) Part i) Scaled Map of Subtracted Product “RF” Total size 450 bp 400 0 Not to Scale Not to Scale To Scale Vector Vector Mark on vector restriction sites used in digest Sac I Eco RI Sac I digest from Table 1: 3,050 bp / 400 bp bands Sac I Sac I Eco RI digest from Table 1: 3,300 bp / 150 bp bands Eco RI Eco RI Draw Composite Map Sac I Eco RI Sac I Eco RI

3. 0 1,000 1,350 500 A) Part ii) Scaled Map of 5’ RACE Product - AF Total size 1,350 bp 5’ RACE Mark on known vector sites Bam HI Sac I Pst I Eco RI Use guide lines or graph paper Sac I digest 4,300/50 Sac I Sac I Eco RI 4,050/300 Eco RI Bam HI 3,850/5,000 Bam HI Bam HI Pst I 4,150/200 Pst I Pst I Composite Bam HI Sac I Eco RI Pst I Pst I Sac I Bam HI Eco RI

4. A) Part iii) Scaled Map of 3’ RACE Product - RB Total size 1,400 bp Pst I Eco RI Bam HI Sac I Mark on known vector sites Kpn I Sac I digest 4,000, 400 Sac I Sac I Eco RI digest 4,250/150 Eco RI Eco RI Bam HI 3,900/500 Bam HI Bam HI Kpn I Pst I 3,800/600 Pst I Kpn I 3,150/700/550 Show both possibilities Kpn I Kpn I Kpn I Kpn I Show double digest Kpn I/Eco RI 3,150/550/150 550 Eco RI 150 550 3,150 Eco RI Kpn I Sac I Bam HI Eco RI Kpn I Kpn I Pst I Sac I Pst I Bam HI Eco RI Kpn I

5. B) Draw a scaled Map of the complete cDNA Using Fig. 1 Map overlay fragments: look for common sites 1,500 2,000 2,300 0 1,000 500 Kpn I Pst I Bam HI Kpn I Eco RI Sac I RB fragment Sac I Eco RI RF fragment Sac I Pst I Bam HI Eco RI AF fragment (reversed) Complete cDNA Sac I Kpn I Pst I Bam HI Pst I Bam HI Eco RI Kpn I

6. C) Sequencing Strategy : Construct / Primer “RF”/T7 RF/SP6 AF/SP6 Pst I deletion of AF/SP6 Bam HI deletion of AF/SP6 RB/SP6 Sac I detection RB/ T7 Pst I fragment of RB recloned / T7 or SP6