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For the dye migration data that had been provided we would get a graph/chart as found below:

Time in minutes
Distance in cm run by Blue Dye
Distance in cm run by Purple Dye
20
0.5
1.2
40
0.7
2
60
1.2
3.4
80
1.7
4.3
 

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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 1.8 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 increase

4 Kb

with 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 proteinase

1 ggcacgaggc caaatatggc gttgaatcac atgtccttga ccactctctt tcttttgatt
61 ggtgcctcta tcattgtttt acaggttgtt gcagaacaac caatatccca agctaaagcg
121 gaatctgcaa tccttcagga ctcaatcgtt aaacaggtta atgaaaatga aaaagctgga

Dra I

 
181 tggaaagctg cactgaaccc tcgattctca aatttcacgg tttcccaatt taagcgcctt
241 cttggagtta agcctacaag aaagggtgat ttaaagggca ttcctatttt aactcatcca

Eco RI

 
301 aaacttttgg agttgccaca agagtttgat gcacgagtgg cttggtctaa ctgtagcact

Pvu I

 
361 attgggagaa ttcttgatca gggacactgt ggttcttgtt gggcttttgg tgctgttgag
421 tcactgtccg atcgtttctg tattcattat ggcttgaata tctctctgtc agcaaatgat

Sph I

 
481 ctctatgcat gctgtggctt tttatgtggg gatggttgtg atggtggata tccgttacaa
541 gcttggaagt actttgtccg caagggtgtg gtcacagatg agtgtgaccc ttactttgat

Eco RV

 
601 aacgagggat gttcccaccc tggatgtgaa cctgcatatc ccaccccaaa gtgtcacagg
661 aagtgcgtta aacagaattt actctggagt aggtccaagc attttggcgt caatgcatac

Hind III

 
721 atgatcagct ccgatccaca cagtatcatg acagaagtgt acaagaatgg accagttgag
781 gtctctttta ctgtttacga ggatttcgct cactacaagt ctggagttta caagcatgta
841 actggtgata taatgggagg ccatgctgtt aaactcatcg gatggggaac ctccgaggat
901 ggagaggact attggcttct tgccaatcag tggaacagag gctggggtga tgatggttac
961 tttaagatca gaagaggaac aaacgaatgt gaaatcgaag atgaagtggt tgcaggattg
1021 ccttcagcca gaaatctgaa cgtggaactt gatgtctctg atgctttcct tgacgccgca
1081 atgtgaatgc taattcttca accaaacgct aaatagaatg caagtttctg aagactgggg
1141 aacaataaca tattaagttt attattcttc ccctcttaag ttgtctgcaa ttgttccact
1201 atcttattat cagtgaagtg ctttccttat ctgttatgta tatcttcaca agtactttgt
1261 caatcttggc actgttgtaa cattaatatg aattttactt aacattatca atgaactgta
1321 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 mm
Size of DNA in Kbp
35
6.37
40
4.82
45
3.67
56
2.32
61
1.93
71
1.26
85
0.7
110
0.22
 

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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