DNA Isolation Report

Summary Table

The expected yield is calculated by using the following formula:

Concentration (μg/ml) = (A260 reading – A320 reading) x dilution factor x (A260 reading x 50 μg/ml)

i.e. Concentration = (1.152-0.026) x 20 x (1.152 x 50) = 1297.152

This is then multiplied by the sample volume to give the DNA yield:

DNA yield = 1297μg/ml x 1ml = 1.297mg expected yield

220 230 240 250 260 270 280 290 300 310 320 330 340 3500

0.5

1

1.5

Absorbance of DNA

Nanometers

Ab

sorb

ance

The DNA sample contained slight RNA contamination with an A260:A280 ratio of 2.106. The yield of our DNA sample was less than the expected yield due to this contamination.

Protocol

Final Volume DNA 1A260 1.152A280 0.547

A260:A280 2.11

PurityRNA

contaminationDilution Factor 20

Diluted DNA (μg/ml) 57.6Original DNA

(mg/ml) 1.152Yield (mg) 1.152

Yield (mg/g Wet Cells) 2.304

Expected Yield (mg) 1.297

Original: Starting Volume 5 mL, 25% SDS New: Starting Volume 10 mL, 12% SDS

Cell Lysis Add 0.4 mL 25% SDS to 5 mL E. coli

cell suspension, mix well Incubate at 65°C for 10 min

Protein Removal Add 1.3mL 5 M NaPerchlorate, mix

well Add 6.7 mL chloroform in the fume

hood and shake with a mechanical wrist arm shaker for 20 min at room temperature

Cell Lysis Add 2 mL 12% SDS to 5 mL E. coli

cell suspension, mix well Incubate at 65°C for 10 min

Protein Removal Add 3mL 5 M NaPerchlorate, mix

well Add 15 mL chloroform in the fume

hood and shake with a mechanical wrist arm shaker for 20 min at room temperature

Removing Chloroform: Centrifuge and take off the bottom

chloroform layer Carefully layer on 2 volumes of ice-

cold 100% ethanol to the aqueous (top) layer

Removing Chloroform: Centrifuge and take off the bottom

chloroform layer Carefully layer on 2 volumes of ice-

cold 100% ethanol to the aqueous (top) layer

Collect the Nucleic Acid which precipitates at the interface by twirling a glass rod

Collect the Nucleic Acid which precipitates at the interface by twirling a glass rod

Dip the nucleic acid laden rod into 70% ethanol to dissolve excess salts from the nucleic acid

Dip the nucleic acid laden rod into 70% ethanol to dissolve excess salts from the nucleic acid

Dissolve the nucleic acid in 1.0 mL T.E Dissolve the nucleic acid in 2.0 mL T.E

Obtain a UV spectrum of the preparation after appropriate dilution (usually at least 1 in 20)

Reserve 2 x 200μL aliquots for gel analysis

To one of the samples add Ribonuclease (20μL of 200 μg/mL RNase)

Store both samples at 4°C

Obtain a UV spectrum of the preparation after appropriate dilution (usually at least 1 in 20)

Reserve 2 x 200μL aliquots for gel analysis

To one of the samples add Ribonuclease (20μL of 200 μg/mL RNase)

Store both samples at 4°C

The method remains the same because the steps taken to extract the DNA in both scenarios are identical and each step must be carried out to certain ratios. However, the protocol reveals the exact measurements and concentrations used and so the protocols will differ depending on the starting volumes of E. Coli and the concentration of SDS. All other added materials will differ in turn in order to maintain these important ratios.

Jonathan Diab (440 247 241) - 2ThuY