Genomic Dna

Genomic DNA post Lab analysis : goal is to isolate pure genomic dna from e.coli to be used next time to amplify the phoA gene using PCR

Use CER framework for the analysis and make sure to answer the following questions:

1.Correctly classified alkaline phosphatase and described the reaction it catalyzes.

2.Accurately describes the overall goal of the project and how isolating genomic DNA fits into achieving the goal.

3.The relationship between absorbance and concentration was correctly described. Correctly made conclusions on whether their A260 readings followed this relationship.

Explained whether they had to omit any A260 readings and why.

4.Correctly assessed the accuracy of the average concentration of isolated DNA using their data and answer to question 3.

5.The purity assessment of the isolated DNA was correct, the contaminate was correctly identified, and the student used their data to justify their conclusion.

6.Correctly identified one step whose failure could lead to the contamination identified in question 5 or if the DNA was pure correctly described a step that could fail and lead to an identified type of contamination.

7.Correctly explains how RNA and protein contamination affect the calculated concentration of DNA. Explains if the calculated concentration is unaffected, increased or decreased and how contamination causes that change or no change.

8.Correctly made a final assessment of the accuracy of the average concentration of isolated DNA using all data. Identified how an inaccurate concentration and contamination will affect the next step in the project.

9.Table is included. This includes a descriptive title. All data presented in the table is calculated correctly. All calculations are included in the appendix.

Additional info:

Bacteria used: E.coli strain HB101

A: Isolation of genomic dna

Reagents : saline-edta, lysozyme, RNAseA, 5M sodium perchlorate, 20% SDS, Chloroform-isoamyl alcohol (24:1), 100% EtOH, DI water.

B. Quantification of nucleic acids : I prepared 3 dilutions of the isolated dna sample as shown below;

Sample ,dH2O, DNa sample, Final volume, Dilution factor

blank ,1000 l , 0l, 1000l , N/A

A , 900 l, 100l , 1000l, 10

B, 980 l, 20l , 1000l , 50

C , 990 l , 10l , 1000l , 100

C. Collected data: We used genesys spec to take absorbance readings at 260nm and 280nm for each dilution sample:

sample, Dilution factor, A260, A280, Concentration of dna in the cuvette , Concentration of isolated DNA, A260/A280 ratio

A, 10, 1.497, 0.781, A260X50(dsDNA), Dna in the curvette X Dilution factor, 1.917

B, 50, 0.235, 0.113 , “, ” “, 2.08

C , 100, 0.116, 0.051, ,” “, 2.27

Average DNA concentration and average a260/a280 ratio: ?

Additional note: we were aiming for a pure isolated genomic dna – 1.8, my A260/A280 ratio is too high suggesting that mine is impure and contaminated with RNA pls note the reason where it could have gone wrong