Seat Number B10

Table 1. Comparison and contrast in the procedures of DNA isolation between plants, animals, bacteria, and yeast.

Source
Category or Step
Plants Animals Bacteria Yeast

Materials needed Cetyltrimethylammoniu Extraction buffer, Tris base, proteinase K, YPD, centrifuge, Harju
m bromide (CTAB) proteinase K, eppendorf phenol/chloroform, 200 buffer, ice-ethanol,
buffer, centrifuge, tubes, centrifuge, proof ethanol, RNAse, vortex, chloroform,
isopropanol, 70% ammonium acetate, ethanol, SDS, EDTA, Pasteur pipette, 70%
ethanol, 2 mL centrifuge isopropanol, 70% tryptone, yeast extract, ethanol, vacuum dryer,
tubes, SpeedVac, TE ethanol, TE buffer, NaCl, LB medium, TE TE buffer, RNAse
buffer, chloroform RNAse, lysis solution, buffer, Lysis buffer, cocktail
column preparation vortex, centrifuge,
solution eppendorf tubes,
incubator

Preparation of DNA Chill plant samples in On your animal tissue After growing an ​E. coli After growing a yeast
source liquid nitrogen, then samples, add liquid culture in LB medium culture in YPD
afterwards, grind the nitrogen. Using a mortar overnight, transfer it on overnight, transfer it on a
plant samples using a and pestle, grind the an eppendorf tube and microcentrifuge tube.
mortar and pestle (doing samples for for 1 minute. centrifuge at maximum Centrifuge for 1-5
this in room temperature speed for 1 minute. minutes.
is fine). The aim is to
powderize the plant
samples to get optimal
DNA extracts.

Lysis Add CTAB extraction Add lysis solution, then Get rid of the Add the Harju buffer
 buffer on the sample, add proteinase K. Then supernatant liquid, and and subject the tubes for
then mix and vortex. For vortex it. This must be put lysis buffer on the 2 minutes in a dry
30 minutes, put the incubated for at least 2-4 cell pellet, then vortex. ice-ethanol bath. Then,
suspension in a warm hours at 55 ​°C
​ . Add lysis Incubate for 1 hour at 37 repeat this process again,
bath. Centrifuge for 5 solution, then vortex. °​C. Add chloroform and and vortex for 30
minutes. Add RNAse Incubate at 70 °C for 10 invert the tube to get a seconds. Before
solution and incubate. minutes. Add column homogenate. Centrifuge, vortexing for 2 minutes,
Add chloroform. Vortex preparation solution and a white aqueous add chloroform,
then centrifuge. Repeat then cortex. (protein) layer will afterwards centrifuge for
the previous 2 steps in a appear. Remove it and 3 minutes. In a tube
new tube until there will repeat the steps until no containing 100% ethanol,
be a clear upper phase. more white upper layer transfer the aqueous
appears.   phase. Incubate for 5
minutes then centrifuge
for another 5 minutes.

Precipitation Get the clear upper Add ethanol and vortex. Add cold 200 proof Using a Pasteur pipette,
phase and add cold Decant the supernatant. ethanol and gently mix. get decant the super-
isopropanol, then Make sure that there are For at least 30 minutes, natant liquid. Then,
incubate at -20 ​°C
​ for 15 no ethanol left. incubate at -20 ​°​C. using 70% ethanol, wash
minutes. Centrifuge for Centrifuge to make sure. Centrifuge at 4 ​°​C for 15 the pellet. Centrifuge for
then remove the minutes. Decant 5 minutes in room
supernatant. Wash with supernatant and wash temperature, and decant
70% ethanol and decant. with 70% ethanol. the supernatant. Air-dry
Dry in a vacuum dryer. Centrifuge, then air-dry. in room temp.

Purification and Be careful not to dry the Add an elution solution Using TE buffer, you Add pellets in a TE
dissolution DNA. Warm the pellet, to elute DNA. Incubate may resuspend the DNA. buffer or water.
then using the TE buffer, for 5 minutes, then
dissolve the DNA. centrifuge for 1 minute.