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Protocol.: Original: Starting Volume 5 ML, 25 % SDS New: Starting Volume 20 ML, 10% SDS Cell Lysis

This document provides a protocol for isolating DNA from E. coli cells. It involves lysing the cells with SDS, removing proteins with sodium perchlorate and chloroform extraction, precipitating the DNA with ethanol, and dissolving it in TE buffer. The protocol is modified for a larger starting volume of E. coli suspension and lower SDS concentration. Quality checks are performed with UV spectroscopy and agarose gel electrophoresis.

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Phi Nguyen
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67 views3 pages

Protocol.: Original: Starting Volume 5 ML, 25 % SDS New: Starting Volume 20 ML, 10% SDS Cell Lysis

This document provides a protocol for isolating DNA from E. coli cells. It involves lysing the cells with SDS, removing proteins with sodium perchlorate and chloroform extraction, precipitating the DNA with ethanol, and dissolving it in TE buffer. The protocol is modified for a larger starting volume of E. coli suspension and lower SDS concentration. Quality checks are performed with UV spectroscopy and agarose gel electrophoresis.

Uploaded by

Phi Nguyen
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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0.

4 Isolation
ml 25% SDS
DNA
Protocol

Protocol.

ml 10% SDS

Put in your new starting volume and SDS

concentration

Original: Starting Volume 5 mL, 25 %


SDS

New: Starting Volume


SDS

20 mL, 10%

Cell Lysis
Add 2 mL
20 ml of E. coli suspension
Add 0.4 ml
10% SDS,
25 % SDS to
to 10 mL
5 mL E. coli
E.coli cell
cell
suspension, mix well.
suspension,
Incubate at 65 oC for 10 min.
mix well.
Protein Removal
Incubate at
Aqueous layer, contains nucleic
Aqueous
acid acidlayer, contains
nucleic acidacidAdd 1.3 mL 5 M
65oC for 10
NaPerchlorate, mix well
min.
Add () mL chloroform in the fume
hood and shake with a mechanical
Protein Removal
Add 1.3 mL 5 M NaPerchlorate, mix well wrist arm shaker for 20 mins at
Add 6.7 mL chloroform in the fume hood room temperature
and shake with a mechanical wrist arm
denatured
Chloroform
proteins
layer, contains denatured proteins
shaker Chloroform
for 20 min atlayer,
roomcontains
temperature
5 ml of E. coli suspension Cell Lysis

Removing Chloroform:

Centrifuge and take off the bottom


chloroform layer
Carefully layer on 2 volumes of icecold 100 % ethanol to the aqueous
(top) layer

Removing Chloroform:
Centrifuge and take off the bottom
chloroform layer
Carefully layer on 2 volumes of icecold 100% ethanol to the aqueous
(top) layer.

DNA Isolation Protocol

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 2.0 mL T.E.

Dissolve the nucleic acid in 2.0 mL T.E.

DNA Isolation Protocol

Obtain a UV spectrum of the


preparation after appropriate dilution
(usually at least 1 in 20)
Reserve 2 X 200 L aliquots for gel
analysis.
To one of the samples add
Ribonuclease (20 l of 200 g/ml
RNase)
Store both samples at 4oC until the
next lab session.

Obtain a UV spectrum of the


preparation after appropriate dilution
(usually at least 1 in 20)
Reserve 2 X 200 L aliquots for gel
analysis.
To one of the samples add
Ribonuclease (20 l of 200 g/ml
RNase)
Store both samples at 4oC until the
next lab session.

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