Homework 3: Recombinant DNA and RFLP Analysis 10 points due 10/17/11

name _______________________________

Part I: Recombinant DNA and Molecular Cloning The first part of this assignment will involve answering questions concerning 1) cutting of the following DNAs with the restriction enzyme, EcoRI 2) ligating together of the resulting fragments 3) transformation of bacteria with the resulting ligation products and 4) identification of the transformants on selective media. Note, ApR means ampicillin resistance

EcoRI
0.7 kb 1.5 kb

EcoRI
1.0 kb

1. (0.5 point) You cut the linear DNA and the pUC19 vector with EcoRI. Assume that all cutting is complete (that all copies of the linear and vector DNA are cut at all EcoRI cut sites). What are the 4 sizes of fragments that result? (you can round the vector size to the nearest kb = kilobase)

2. (2 points) You add the fragments together with the ligase enzyme. (0.5 point) Which two of the four fragments would not be included in any circular ligation products? Why is this?

(1.5 points) In the space below, draw diagrams depicting three circular ligation products. Include the sizes of each of the ligated pieces in each. Also label each with the total size of the circular, ligated DNA.

3. (1.5 points) You transform bacteria with the ligation products. The bacteria are then grown on LB + ampicillin + X-gal. Fill in the following table describing the colonies that result from transformation of bacteria by each of the recombinant DNAs. Total size of ligated DNA Grows on ampicillin? (Y/N) Blue or White? (B/W)

4. (0.5 point) Mistakes often occur in molecular cloning. If, after transforming the bacteria with ligation products, the plate is covered with a bacterial lawn, which one of the following errors has likely occurred? error a. Forgot to add the ampicillin to the plates. b. Forgot to add the X-gal to the plates. c. Forgot to add the restriction enzyme to the digests.

Dont forget Part 2 on the next page!!!!

Part 2: RFLP Analysis

You perform restriction digests on three DNAs and separate the fragments resulting from each, using agarose gel electrophoresis. The DNA sequences that you cut are shown below: 5 GGCTCTAGCTTTACGCCAGTTCCGAATTAGTTCGGCGGCCT 3 3 CCGAGATCGAAATGCGGTCAAGGCTTAATCAAGCCGCCGGA 5 5 GGCTCTAGCTTTACGCCAGTTCCGAATTAGCTCGGCGGCCT 3 3 CCGAGATCGAAATGCGGTCAAGGCTTAATCGAGCCGCCGGA 5 5 GGCTCTAGGTTTACGCCAGTTCCGAATTAGTTCGGCGGCCT 3 3 CCGAGATCCAAATGCGGTCAAGGCTTAATCAAGCCGCCGGA 5 original sequence mutation 1 mutation 2

You cut the sequences using the restriction enzyme AluI. This enzyme is a blunt end cutter that cuts at the sequence 5AGCT3 as shown below 5AGCT3 3TCGA5 5AG 3TC CT3 GA5

(1.5 points) Fill in the following table with the fragments and their sizes that result from cutting the DNA with the AluI enzyme. Sequence original Mutation 1 Mutation 2 (3 points) The results of your electrophoresis of the cut DNA are shown below. The lanes on the gel have been labeled with the sample run in each (Orig, Mut1, Mut2). In the spaces along the side of the gel, note the size, in base pairs, of the fragment(s) each lines up with. (only one size per space)
Orig Mut 1 Mut 2 size (in base pairs)

# of fragments

Fragment lengths (in base pairs)

You transfer the DNA from the gel to nylon and probe it using the following DNA probe: (note that the probe is labeled with radioactivity and that both the DNA on the blot and the probe are denatured prior to hybridization) 5 TCCGAATTAG 3 3 AGGCTTAATC 5 (1 point) Circle the bands on the gel above that will appear in the autoradiogram that results.