Question

Please I need help on questions 1-4 in great detail please

Load 15 mu l of the following samples from the above section onto the simple Wells. Seal the wells with agarose and electrophorese until the bromophenol blue in the samples has migrated to within 2 mm of the positive electrode end of the gel. Remove the gels from the unit and stain them as described in Section IV. Measure the distance of the DNA bands (in cm) from the sample wells and draw a picture of the DNA bands in each gel lane. Note the relative intensity and positions of the DNA bands in lane 3 and lane 4. Note especially the intensity of the next to the largest DNA band. The size of the DNA bands in lanes 2 and 6 should be 21.2, 7.4, 5.8, 5.6, 4.8 and 3.5 kilobase pairs (see Figure 2-2). From the map of phage lambda given in Figure 2-2, identify the DNA bands that contain the genes for the head proteins and the genes that control lysis of the host cell. Maps of the restriction sites for EcoR1 and Bam H1 in lambda DNA are given in Figure 2-2. With this information: A. Calculate the length of DNA fragments that should have been produced when lambda DNA was digested with both EcoR1 and BamH1. List these values below and indicate the two fragments that contain the cohesive ends. B. Identify the above fragments on lanes 3 and 4 of your gels. A comparison of the results of lanes 3 and 4 on your gels can be used to identify the DNA terminal fragments of phage lambda without acknowledge of the map shown in Figure 2-2. Explain. In DNA of uniform composition, a specific hexanucleotide occurs by chance once in every 4016 base-pairs. The recognition sequence for EcoR1 is a hexanucleotide (see Part A-1, Table 1). How many restriction sites for EcoR1 would you expect to exist in lambda DNA if the phage DNA were of uniform composition? There are few restriction sites for a number of enzymes including EcoR1 and Hpal in the left 20 kilobase of lambda DNA. Explain. A. Genetic Map of Phage Lambda B. Restriction Map of Lambda DNA - EcoR1 Arrows indicate the position of restriction sites for EcoR1. Sites are in base-pairs from the left end of the phage DNA. Lambda DNA is 48, 502 base-pairs long. C. Restriction Map of DNA - Bam H1 Arrows indicate the position of restriction sites for Bam H1 Sites are in base-pairs from the left end of the phage DNA.

Answer 1

1.Seeing the restriction map of Lambda phage and the Eco R1 restriction sites, the bands that contain genes for head proteins and genes for lysis would be the 1st and the last band respectively, in well no 2. That is the first thick band (21226 bp) and last band (3630 bp)

2 A )The fragments produced when Lambda phage is cut with both Eco R1 and Bam H1 can be calculated by seeing the restriction pattern of both the enzymes and combining both, since they are very specific.Base pairs will be different as they will have to be subtracted from one another

1.) 5505 bp, 2) 15721 bp 3) 1120 bp 4) 3758 bp 5) 1868 bp 6) 3775 bp 7) 2752 bp 8) 4669 bp 9) 2564 bp 10) 3240 bp 11) 3528 bp. Total of these basepairs come to 48500 bp ,as of length of Lambda phage.The 15721 fragment and the 3758 fragment cut by Eco R1 would have cohesive ends

B)

4. Restriction enzymes are highly specific about the region they bind, to cut at that site. The GC rich area in Lambda phage corresponds to Pallindrome sequences that contain around six base pairs. Pallindrome sequence reads same in both forward and backward direction. Example 5' to 3' may read GAATTC .The complimentary bases will read CTTAAG on the opposite strand, which is the same as the other strand backwards. There would be 5 restriction sites for Eco R1 on Lambda DNA.