A.

Experiment 1
1. Objectives
To isolate genomic DNA from Escherichia coli
2. Materials

a. Tris base (Calbiochem-Behring)

b. Proteinase K (Sigma-Aldrich)
c. Phenol\chloroform (1: 1) (EM Science)
d. 200 proof ethanol (Pharmco-AAPER)
e. RNAase (Life Technologies, Invitrogen™)
f. Ethanol
g. SDS
h. EDTA
i. Tryptone
j. Yeast extract
k. NaCl
l. LB medium (see Recipes)
m. TE buffer (see Recipes)
n. Lysis buffer (see Recipes)

3. Method
A. E. coli Genomic DNA Extraction (Conventional DNA extraction protocol

i. 1.5 mL of the overnight E.coli culture has been transferred to 1.5 mL Eppendorf
tube and centrifuge at max speed for 1 min.
ii. 600μL lysis buffer has been resuspend and vortex to completely resuspend cell
pellet.
iii. Incubated 1 h at 37 degree celcius.
iv. Equal volume of choloroform are added and mixed well by inverting the tube
until completely mixed.
v. The tube has been spun at max speed for 5 min.
vi. The upper aqueous has been transferred to a new tube by using 1 mL pippete.
vii. Those step repeated untill the white protein layer disappears.
viii. 3 volume of 200 ethanol added
ix. The tube has been incubated at -20 degree for 30 min or more.
x. The tube has been spun at max speed for 15 min at 4 degree celcius
xi. The supernatant has been discard and DNA has been rinse with 1mL of 70%
ethanol.
xii. The tube has been spun at max speed for 2 min and let air dry.
xiii. DNA has been resuspended in TE buffer.
xiv. Isolated Gemonic DNA checked on an agarose gel.
 B. E. coli genomic DNA extraction(Kit DNA extraction protocol)

i. Cells have been harvested in 1.5 mL microcentrifuge tube by centrifugation for 1

min at full speed.
ii. Cell peletes in 200 l of Buffer CL has been resuspended completely.
iii. 20 μL Protinase K solution has been added. Has been vortex, and incubated at
56 degree celcius for 15 min. the cell has been spun for removing any drops
from inside of the lid.
iv. 20 μL of Rnase has added , vortexed,and incubated for 5 min.
v. 200 μL of Buffer BL has added to the tube, vortexed, and incubated for 1 min at
70 degree celcius.
vi. 200 μL of absolute ethanol has been added, pulse vortexed, spun down to
remove any drops inside of the lid.
vii. All the mixture transferred, to the SV column, centrifuged for 1 min.
viii. 600 μL of Buffer BW has been added, centrifuged for 1 min.
ix. 700 μL of Buffer TW applied, centrifuged for 1 min and reinserted the SV column
back into the collection tube.
x. At full speed, the tube centrifuged for 1 min to remove residual wash buffer. SV
column placed into a fresh 1.5 mL tube.
xi. 25 μL of sterilized water was added, incubated for 1 min and centrifuged at full
speed for 1 min.

4. Results

As we run in computer application, we have to result, first result is wrongly

technique, so we decide to did it again and, the second trial, the DNA was been
found.

Figure 1 the first trial well no 8 from left

 Figure 2 our DNA in well 7 from left

Size of Genomic DNA for Ecoli in kbp: 4,640,00bp

5. Discussion

As we know, the purpose of DNA extraction is to obtain DNA in a relatively purified

form, which can be used for futher investigating, such as, PCR, etc. The protocol
basicly consist two parts, first, a technique to lyse the cells gently and solubilize the
DNA and Enzymatic or chemical methods to remove contaiminating protiens, RNA,
or macromolecules. As we made in lab, the first part of precipating, we used phenol
denatures proteins and dissolves denatured proteins and cloroform, also a protein
denaturant. We added the salt, to interrupt the hydrogen bonds between the water
and DNA molecules. Then, DNA is then precipitates from the protein in a subsequent
step with isopropanol and ethanol because of, the presenceof cations, ethanol
induces a structural change in DNA molecules that causes then aggregate and
precipitate out of solution. The DNA ispelleted bu spinning with a centrifuge and the
supernatant removed. We rinsed the DNA with 1ml of 70% ethanol is for, washing.
The term washing is refering to remove salts and other water soluble impurities but
not resuspend the DNA. Then, the term resuspension, is referring to ensure stability
and long term storage. The protocol used in Agarose gel to see the quality of DNA, it
is perform or not? By mixing 5μL of DNA with 3μL loading buffer, load this mixture
into a 1% agarose gel. Then, analyze results with the usage of computer appllication.

6. Conclusion

So, the objective has been achived, we made it well. And it will remind us to more
carefull how to handle the instrumen, how to do the techniques as well to prevent
the same mistake to another experiment. The result reported comfirm that the
extraction method, applied to Ecoli, produces DNA of suitable quantity and quality
for subsequent PCR based detection applications.

7. Reference

rice.plantbiology.msu.edu/training/DNA_Extraction_Overview.ppt. achived on
19/1/2018
http://gmo-crl.jrc.ec.europa.eu/summaries/PT73%20TM_DNAExtr_report.pdf.
Achived on 19/1/2018
B. (2.B)
Procedure

1. 3μL of 6× loading dye dispensed onto a strip of parafilm.

2. 7μL of genomic DNA has been added to the loading dye and mixed them.
3. 10μL of digested DNA has been added, to 3 μL of loading dye for digested genomic DNA.
4. The well (far left or far right for DNA marker.ladder) has been loaded with 3.5 μL DNA
ladder.
5. Remaining wells, has been loaded with premixed DNA-loading dye.
6. The electrophorosis tank assembled with electrodes and connected to power pack
7. Electrophorosis has been performed at 70 volts for 30 minutes.
8. The running stopped and the power turned off.
9. The gel observed under UV light using Gel Dic Easy System (BioRad)