Molecular genetics II Essay

 For the dye migration data that had been provided we would get a graph/chart as found below:Time in minutesDistance in cm run by Blue DyeDistance in cm run by Purple Dye200.51.2400.72601.

23.4801.74.3 We know that it is an established fact that the purple dye or bromophenol blue dye runs with double stranded DNA of about 300 bp in length and the blue-green dye or xylene cyanol dye runs with double stranded DNA of about 4 Kb in length on a 1% agarose gel (Refer text: page 4.5). Hence we can easily calculate the distance run by a 300 bp fragment at a specific time point between 0-80 minutes using the purple dye curve and the distance run by a 4 Kb fragment using the blue dye. Thus, Q1. a) in 35 mins 300 bp fragment would have migrated 1.

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8 cm.b) in 55 mins 300 bp fragment would have migrated 3.1 cm. Q2. a) in 45 mins 4 Kb fragment would have migrated 0.8 cm.

b) in 70 mins 4 Kb fragment would have migrated 1.4 cm. Q3. From the data represented by the graph (Figure 4.2) one would be able to calculate distance of migration of DNA of any length between 10 Kb and 100 bp. Thus a 1 Kb DNA fragment runs a) ~ 2.

5 cm on a 1.4% agarose gel.b) ~ 4.

0 cm on a 0.7% agarose gel.   Q4. From the data represented by the graph (Figure 4.2) one would be able to calculate distance of migration of DNA of any length between 10 Kb and 100 bp.

 Thus a 10 Kb DNA fragment runs ~ 1.5 cm on a 0.5% agarose gel.     Q5. When run at a constant voltage, the rate of migration of a 4 Kb DNA fragment would increase4 Kbwith decrease in agarose concentration in the gel. From figure 4.2 we can observe that at a specific point of time the 4 Kb fragment would have run 2 cm, 3 cm, 4 cm and 5 cm when the agarose concentration was 1.4%, 1.

2%, 0.9%, 0.7% and 0.5% respectively.Q6.

N.rustica cDNA for cathepsin B-like cysteine proteinase1 ggcacgaggc caaatatggc gttgaatcac atgtccttga ccactctctt tcttttgatt61 ggtgcctcta tcattgtttt acaggttgtt gcagaacaac caatatccca agctaaagcg121 gaatctgcaa tccttcagga ctcaatcgtt aaacaggtta atgaaaatga aaaagctggaDra I 181 tggaaagctg cactgaaccc tcgattctca aatttcacgg tttcccaatt taagcgcctt241 cttggagtta agcctacaag aaagggtgat ttaaagggca ttcctatttt aactcatccaEco RI 301 aaacttttgg agttgccaca agagtttgat gcacgagtgg cttggtctaa ctgtagcactPvu I 361 attgggagaa ttcttgatca gggacactgt ggttcttgtt gggcttttgg tgctgttgag421 tcactgtccg atcgtttctg tattcattat ggcttgaata tctctctgtc agcaaatgatSph I 481 ctctatgcat gctgtggctt tttatgtggg gatggttgtg atggtggata tccgttacaa541 gcttggaagt actttgtccg caagggtgtg gtcacagatg agtgtgaccc ttactttgatEco RV 601 aacgagggat gttcccaccc tggatgtgaa cctgcatatc ccaccccaaa gtgtcacagg661 aagtgcgtta aacagaattt actctggagt aggtccaagc attttggcgt caatgcatacHind III 721 atgatcagct ccgatccaca cagtatcatg acagaagtgt acaagaatgg accagttgag781 gtctctttta ctgtttacga ggatttcgct cactacaagt ctggagttta caagcatgta841 actggtgata taatgggagg ccatgctgtt aaactcatcg gatggggaac ctccgaggat901 ggagaggact attggcttct tgccaatcag tggaacagag gctggggtga tgatggttac961 tttaagatca gaagaggaac aaacgaatgt gaaatcgaag atgaagtggt tgcaggattg1021 ccttcagcca gaaatctgaa cgtggaactt gatgtctctg atgctttcct tgacgccgca1081 atgtgaatgc taattcttca accaaacgct aaatagaatg caagtttctg aagactgggg1141 aacaataaca tattaagttt attattcttc ccctcttaag ttgtctgcaa ttgttccact1201 atcttattat cagtgaagtg ctttccttat ctgttatgta tatcttcaca agtactttgt1261 caatcttggc actgttgtaa cattaatatg aattttactt aacattatca atgaactgta1321 ttattgtggt c Q7. On digesting N.rustica cDNA for cathepsin B-like cysteine proteinase (of length 1331 bp) with  1.      Eco RI: two fragments of sizes 963 bp and 368 bp are produced.

2.      Pvu I: two fragments of sizes 899 bp and 432 bp are produced.3.      SphI: two fragments of sizes 840 bp and 491 bp are produced.4.      Eco RV: two fragments of sizes 802 bp and 529 bp are produced.5.      Hind III: two fragments of sizes 792 bp and 539 bp are produced.

6.      XbaI does not cut the cDNA since the corresponding restriction site is absent. ANALYSIS OF PLASMID DNA AFTER DIGESTION WITH RESTRICTION ENZYMES Q8. Digesting pCathB with Bam HI will result in the linearization of the plasmid and thus a single fragment of 4.29 Kb is produced. This is due to the presence of a lone Bam HI restriction site in the polycloning/polylinker site. Q9. XbaI and KpnI restriction sites are present at the extreme ends of the polylinker site.

Hence on digesting pCathB with Xba I and Kpn I enzymes 2 fragments of sizes 2.89 Kb and 1.40 Kb are obtained. This is the result of the addition of the 72 bp polylinker site to the cathepsin B cDNA from the plasmid backbone.

 Q10. Two fragments of sizes 3.75 Kb and 540 bp are obtained when pCathB is cut with Hind III which targets the corresponding restriction sites present one each on the cathepsin cDNA insert and the polylinker site of the plasmid.

 Q11. When pCathB is cut with Hind III and Bam HI three fragments are obtained. The plasmid backbone of size 2.96 Kb and the two fragments of cathepsin cDNA of sizes 790 and 540 bp are the three fragments. They are produced out of the cuts made at the Bam HI restriction site on the polylinker and the two Hind III sites present one each on the polylinker and the cDNA. Q12.

Since there is only one EcoRV site in the cDNA and since the polylinker Eco RV sites are lost on ligation, digesting pCathB with EcoRV enzyme results in the linearized form of size 4.29 Kb. Q13. Cutting pCathB with Xba I and Bam HI also results in linearization and hence a single fragment of size 4.29 Kb.

This is because of the fact that Bam HI and Xba I sites are absent in the cDNA and both are present on the 5’ region of the polylinker site.Figure 2. ? –BstEII Ladder Distance migrated through the Agarose Gel from the point of Origin in mmSize of DNA in Kbp356.

37404.82453.67562.32611.93711.26850.

71100.22 The semi logarithmic graph shows a straight line of best fit through the points:  A schematic diagram representing the pattern of migration of different fragments of the ?- Bst EII digest on a 1% Agarose Gel:Figure3. Depiction of Agarose Gel after digestion of pCathB with various restriction enzymes and analysis by gel electrophoresis

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