PRACTICAL 1

ISOLATION OF PLASMID BY ALKALINE LYSIS AND PHENOL PROTEIN EXTRACTION AND DNA
PRECIPITATION

INTRODUCTION

Scientist from many different disciplines have used the technique of genetic engineering to genetically
alter the bacteria and eukaryotic organism. In genetic engineering, the genes are located from one
organism and incorporated by manipulation in a laboratory into other bacterial or eukaryotic cells. The
first step in genetic engineering is the extraction of DNA from the cell that possess the desired gene. To
extract DNA from bacteria, the cells can be disrupted by chemical or mechanical means or by osmotic
pressure. Cell debris is precipitated with SDS (sodium dodecyl sulfate). The solution 111 (3M kalium 5M
acetate pH 4.8) denatures proteins such as DNase and stabilizes the DNA by forming an K+ shell around
the negatively charged phosphates of the DNA. The DNA is precipitated as a viscous, translucent mass
with cold alcohol.

METHOD

1) mL of overnight culture was transferred into centrifuge tube and had been centrifuged ata 12000
rpm for 1 min.
2) the supernatant was removed until the pellet dry.
3) 100 micro litre of ice cold solution was added
4) And been eft for 5 min in ice
5) 200 micro litre of solution II was added. The tube was tightly closed and the content mixed by
inverting the tube rapidly for a several times. The tube is placed in ice for 5 min.
6) 150 micro litre of ice cold solution III was added. The content was mixed by inverting the tube
raidly for a several times and stored in ice for 5 min.
7) Then centrifuged at 12000rpm for 5 min
8) The supernatant was transferred to a fresh tube.
9) Next step is purified DNA from supernatant using phenol protein extraction (part2)
10) The purified DNA should be purified further using phenol precipitation (part 3)

2. ISOLATION OF DNA BY OHENOL PROTEIN EXTRACTION

1) An equal volume of absolute phenol-chloroform-isoamyl alcohol was added to the DNA samples
in a fume hood.
2) Mixed by gentle inversion or vortex for 10 sec with lid closed tightly. (phenol is highly toxic)
3) Then mixture was centrifuged at room temperature for 15 sec.
 4) The aqueous phase should be carefully removed and transferred into a new microcentrifuge tube.
Try to avoid transferring any of the denatured protein tha is located at the interface of the phases.

3.0 ETHANOL PRECIPATION OF DNA

1) 2 volumes (800 micro litre) of 10% cold ethanol ethanol (-20 C) was added into the sample (into
microfuge tube)
2) Mixture was vortexed and incubated the tube at -20C for overnight or -70C for 30 min or more.

RESULT
DISCUSSION

According to the standard quantification of DNA, the DNA can be detected its presence by using bio
photometer that using the UV absorption of 260nm. The concentration of the solution of DNA can be
measured by absorbance at 260nm. According to the result getting from the experiment, the result shows
the ratio of A260/A280 is 1.12.

The ratio shows different value if there are contaminants.

If A260/A280 value is around 1.5, it means the solution contain pure DNA. If A260/A280 value is around
2.0, the solution is determined to having the pure RNA within. If A260/A280 value is around below value
of 1.5 meaning that the solution was contaminated with protein or phenol and lastly If A260/A280 value
is higher than 2.0, it stated that the solution was contaminated with RNA.

Since the experimented value is 1.12, meaning that, the experimented solution had been contaminated
with protein or phenol.

There must be some error occur during experiment is done. The reasons why the experiment lead to
inaccurate result is the sample did not mixed very well with the chloroform resulting the phenol hard to
dissolves on the chloroform. So the precaution step is to repeat the same procedure starting on adding
chloroform alone and be sure no to forget to mix the sample with chloroform so that any trace of phenol
dissolves on the chloroform.

Next, the transferring of the supernatant into another tube did not done carefully. So be very careful while
transfering supernatant to new tube as chloroform contamination is relatively easy to spot as small
droplets precipitating to tube's bottom (Luis, 2016).

The result could be leaded to error due to the handling of the cuvette itself during using biophotometer.
The clear cuvette should be facing the UV light in order to calculate the ratio. Also, Air bubbles or other
visible particles in the cuvette must be avoided. And one thing that is important before using
Biophotometer is before transferring the sample into the cuvette, the solution should be vortex briefly
again prior to measurement to prevent concentration fluctuation.

Next, maybe it’s due to the measuring medium, The absorption behavior of nucleic acids is influenced by
the pH value and the ionic strength of the buffer. One can thus only obtain precise concentrations under
controlled pH conditions and using solutions with low ionic strengths. Because water is not pH-stable,
fluctuating measurement results may occur. Some buffers may exhibit self-absorption in the UV range. In
order to avoid inaccuracies, use the same buffer in which the sample was resuspended/eluted following
isolation for the blank and the sample (Kaeppler-Hanno, Armbrecht-Ihle, & Kubasch, 2015)

REFERENCES

Luis Antonio Ochoa-Ramírez. (2016). How can I remove phenol contamination from DNA sample?
Retrieved November 22, 2018, from
https://www.researchgate.net/post/How_can_I_remove_phenol_contamination_from_DNA_sample

Kaeppler-Hanno, K., Armbrecht-Ihle, M., & Kubasch, R. (2015). Troubleshooting Guide for the
Measurement of Nucleic Acids with Eppendorf BioPhotometer ®, (13), 1–11.