experiment 8: isolation of dna from allium cepa

8/16/2019 Experiment 8: Isolation of DNA from Allium cepa

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Isolation and CharacterizationDNA from Allium cepa (white

Ignacio, Richelle Angelika

Macaraeg, Angeline ClaireRivera, NicoSolano, a!lina Aleccia"iernes, #ian Alfonso


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IN'RDC'IN


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NC*+IC ACIDS

N!cleic acids are io-ol.mers, or large ioessential for all known forms of life/ N!cleic ac

incl!de DNA (deo0.rion!cleic acid) and RNA (

acid), are made from monomers known as n!cleot

'here are two -rinci-al t.-es of n!cleic a

(deo0.rion!cleic acid) and RNA (rion!cleic acid)


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N!cleotides

• !rine or -.rimidine asesonded to s!gars (riose ordeo0.riose) , which int!rn are onded to-hos-hate gro!-s PURINE

PYRIMIDINE


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DNA• Deo0.rion!cleic acid

• ol.mer of deo0.rion!cleotides

• Monosaccharide is &1deo0.riose

• Nitrogeno!s ases are adenine, g!anine,c.tosine, 2 th.mine

• Do!le heli0 str!ct!re

• 3 onding etween the nitrogeno!sases in the center of the heli0 hel-shold do!le heli0 together

Figure 1. Retrieved fromhtt-455www/terravivos/com5sec!re5


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DNA& strands of DNA are com-lementar. strands

• Se7!ence of ases on one a!tomaticall.

determines se7!ence of ases on another

& strands of DNA are anti-arallel

• R!n in o--osite directions

• nl. when & strands are anti-arallel can the

ase -airs form 3 onds that hold the

strands together


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Allium cepa

• Allium cepa, the onion (also called !l oncommon onion), is a monocot !lo!s -er

• It is the most widel. c!ltivated s-ecies of t

gen!s Allium


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%8+C'I"+S


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• 'o isolate DNA from Allium cepa

• 'o determine the -rotein and n!cleic acidconcentration . "1"is S-ectrometr. an

calc!late the asorance ratio

• 'o se-arate com-onents of DNA thro!gh

h.drol.sis

• 'o characterize DNA thro!gh vario!s 7!a


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M+'3D*#9


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A/ Isolation of DNA from on

R+*IMINARI+S

:/ 3eat the ti- of a -i-et in a %!nsen ;ame !

end ack !- towards the to- to form a ho

&/ Cool


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50 mL homogenizing

solution

- Heat in a water bath until the

solution reaches 60°C

- Place in a 250 mL Erlenmeyer las! - "emo#e th

mince the on

- %eight 25

A. 50 mL o 60°C

homogenizing solution

%hole onion

B. &ince$ onion

A. 50 mL o 60°C homogenizing

solution an$ B. &ince$ onion

-Put in Erlenmeyer las! an$ stir abath or 5 minutes an$ stir occas

minutes

- '$$ ( tablets o )ul#erize$ cru$e )

minutes o a$$ing the onion+ an$ !

or ,0 minutes more *stir occasio

minutes+


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- mme$iately transer the las! int

minutes an$ swirl gently

- .uic!ly )our the contents into a ble

(5 secon$s

-/ilter the homogenate through ( lay

"esi$ue

*$iscar$+

/iltrate

- &easure the #olume i

gra$uate$ cylin$er - ranser to a 250 mL b

on ice

-ilt the bea!er at a (5

slowly an$ gently )our ice

$own the inner wall o the


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- 'llow the mi3ture to sit

to 4 minutes until the bub

- )ool out as much 7'

the s)ooler in an$ out

layers+ in , $irection only

-e)arate the remainin

s)oole$ 7' an$ air

*7'+

-%eigh the air- $rie$ 7'

'ir- $rie$ 7'

)reci)itate


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%/ Determination of DNA Concentration an

,90 mg 7'

- issol#e in 494 mL aline s

solution

- "ea$ absorbance at 260 nm an$

- etermine the )rotein concentra

aci$ concentration

- Calculate the absorbance rat

solution

7' solution


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C/ Acid 3.drol.sis of DNAsolate$ 7'

- is)erse in ,90 mL o ,& HCl

test tube

- Co#er the test tube with a ma

mi3ture at ,00°C or (5 minute

agitation

- "emo#e the test tube rom th

allow to cool to room tem)erature

- '$$ 295 mL o $istille$ water

- 7eutralize with ,& 7a;H an$

)a)er

- /ilter the hy$rolyzate


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"esi$ue *$iscar$+ /iltrate

- &a!e

o the

$istille$

7' Hy$rolyzate


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D/ Chemical Characterization ofD/: Dische Reaction

,90 mL 7' Hy$rolyzate


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D/& 'est for hos-hate

,90 mL 7' Hy$rolyzate


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- &i3 well an$ then $ilute to ,0 mL

- Let stan$ or ,0 minutes

- ;bser#e the color o the sol

)reci)itate orme$

Colorless solution with

yellow )reci)itate


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D/$ 'est for -!rines5M!re0ide 'est7' Hy$rolyzateuanine or '$enine

solution

- Place in a small e#a)orating $is

- '$$ a ew $ro)s o concentrate$- E#a)orate to $ryness #ery care

in the ume hoo$

?ellow resi$ue

- @)on moistening with ,0 A;

color

- @)on urther heatingB assumes a

- '$$ ew $ro)s o water an$ then

?ellow solution

- E#a)orate

"e$ resi$ue


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D/@ heeler18ohnson 'est5 'est for -.rimid

,90 mL 7' hy$rolyzate


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R+S*'S AND DISCSS


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A/ Isolation of DNA from onion

Grp # Weight of onion (g)Weight of air-dried DNA ppt

(g)

, 259,154 0905(4

2 2594,( 092(55

4 259(,1 09,4:2

( 25965(2 09254

5 259(2(0 09441

6 259,64 095::

2590: 0944

: 259011 09044,

1 2594,2( 0902(


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Consist of $ asic ste-s4

1.Homogenization1 rocess of reaking !- the onion tiss!es to se-arate a

cells

1 Involves the heating and lending of the onion tiss!es

Homogenization solution 1 allows the DNA to e re

its memrane

Sodium dodecyl sulfate or SDS 1 is a detergent t

reaking

A/Isolation of DNA from onio


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Ethylenediaminetetraacetic acid or EDTA 1 ina

DNAses

Sodium chloride and Sodium citrate 1 ne!trali

negative charges of the -hos-hate gro!-s of the D

Heating

1 Softens the -hos-holi-id in the cell mem

denat!res DNAse

'em-erat!re !sed4 B=C (*eads to denat!ra

when the tem-erat!re B=C is e0ceeded)


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Blending

1 !rther reakdown of cell memranes

1 @< seconds onl. in order to not destro. DNA

2. Deproteinization

1 rocess of removal of the -roteins that are associated

and DNA molec!les

1 Involves the adding of the -rotease enz.me -a-ain


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ul!erized papain " A -roteol.tic enz.me which h.

-roteins clinging to the DNA

Filtration

Material !sed4 Cheesecloth

1 sed to Elter o!t other -arts of the cell (cememrane) eFectivel.

1 Removes an. solid material, res!lting in ahomogenate


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#. recipitation

1 -rocess of forcing the DNA to come o!t of sol!tion via ad

cold alcohol into the sol!tion

$ce"cold %&' ethanol 1 !sed eca!se the DNA is no

alcohol

1 the colder the alcohol, the less sol!will e

1 resence of two la.ers

% D t i ti f DNA C t ti


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%/ Determination of DNA Concentration a

GRUP # $%& n' $& n' A*oran+eRatio

Protein('g,')

, 09,:: 09,:6 ,90, 09,2

2 09,16 09,12 ,902 09,2

4 09,,: 09,: 0964 09,

( 090:( 090:2 ,90 090:

5 094( 094:, 091:2 094(


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GRUP # $%& n' $& n' A*oran+eRatio

Protein('g,')

6 09,: 09,: , 09,5

09 ,4( 09,4, ,902 ,90

: 09,0( 09011 ,905, 0901

1 094:6 094:1 0911 092:


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%:/ Determination of -rotein and n!cleconcentration

G" s-ectro-hotometr.

GDNA asors strongl. at &B=

nm

Groteins asor strongl. at

&H= nm

GNomogram

So!rce4 htt-455c-mcnet/col!mia/ed!5de-t5-hsio5tc


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%&/ Asorance Ratio of DNAG#ives the relative

meas!rement of DNA

and -rotein content in

the isolated DNA

GA good 7!alit. DNAsam-le sho!ld give a

val!e of :/H1:/>

Presenc

Pure

n$icatecontam


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C/ Acid 3.drol.sis• 1( H)l

1 Ca!ses dissociation of DNA into itscom-onents

1 Ca!ses de-!rination

• 1( *a+H1 Ne!tralize the e0cess 3Cl

• 1,,-) 'o destro.41 3.drogen onds aove H=C, acids with

-3 J $1 hos-hodiester onds aove >=C, acids

with -3 J &

H/ *+T S0

BASE H/D+- entose of D

not have a &gro!-

- A mono-hosintermediat

form 5267#6 monophosintermedia

- DNA is stah.drol.sis


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De-!rination

So!rce4 Mahato, R/ I/ Biomaterials for Delivery and Targeting of Proteins and Nucleic Ac

%ooks/Jhtt-455ooks/google/com/-hL Accessed :B Ma. &=:B/


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Chemical characterizatioDNA


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'est for Deo0.riose5 Disreaction#R DNA 39DR*9A'+ D+O9

S*(stan

: '!rid white sol!tion Clear dsol

& *ight l!e t!ridsol!tion "iolet

$ hite t!rid with-reci-itate

!r-le t!r

@ *ight l!e sol!tion !r-le

#R DNA 39DR*9A'+ D+O9


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#R DNA 39DR*9A'+ D+O9S*(stan

< Clear light l!e

sol!tion

Dark viole

B %l!e1gra. sol!tionwith s!s-ected white

-articles

Dark viole

P Clear light l!e

sol!tion with t!rid-reci-itate on to-

s!rface

Clear da

sol!

H N/D Slightl. t!r

> Clear sol!tion !r-le


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TEST F+9

sed to detect the

-resence of deo0.riose

'est for deo0.riose5 DiscreactionEA:E*T9

Di-hen.lamine

So!rce4 htt)D


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+S$T$;E ES03T9

%l!e colored li7!id

sol!tion

$*)$3E9

1 Deh.dration o

Deo0.riose

3&S@(format

h.dro0.1

lev!linaldeh.

1 Com-le0ation

Di hen laminSo!rce4 htt)D


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' t f h h t


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'est for -hos-hates#R DNA 39DR*9A'+ 3S3A'

(stan

: N/D N

& Clear colorless sol!tionwith -reci-itate

Clear color

$ Clear colorlesssol!tionQ no -reci-itate

Clear csol!tionQ n

@ Clear colorless sol!tion Clear colorwith .ellow

#R DNA 39DR*9A'+ 3S3A'( t


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(stan

< Clear colorlesssol!tionQ no -reci-itate

Colorless -rec

B '!rid colorlesssol!tionQ no --t

Clear csol!tionQ n

P Clear sol!tion withwhite --t

Colorleswitho!t -

H N/D Clear color

> Clear colorlesssol!tionQ no -reci-itate

Clear csol!tionQ n


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'est for hos-hateTEST F+9

'o determine the

-resence of -hos-hate

EA:E*T9

3N$

3&S@

Ammoni!m mol


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ositi!e result9

.ellow -reci-itate(ammoni!m

-hos-homol.date)

rinciple9

reci-itation hos-hate

3$@ 3N$ :&(N3@)&Mo@ (N3@)$@/:&M

M id ' t5' t f


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M!re0ide 'est5'est for !r#R DNA 39DR*9A'+ #ANIN+

(stan

: N/D Red r

5un


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(stan

@

ater $ryingD

light yellow resi$ue

ater moisteningDlight yellow solution

(nfini*hed)

ater $

yellow

ater more$ color o

ater 2n$

re$ re

ater a$$itio

orange

<

=rown resi$ue

(nfini*hed)

ar! orang

(nfin

#R DNA 39DR*9A'+ #ANIN+


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(stan

B N/D N

P N/D N

H N/D N

> N/D yellow solutioyellow )r

(nfin

f i id


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TEST F+9

resence of -!rines

'est for !rines5M!re0ide EA:E*T9

Concentrated 3

:=? 3


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+S$T$;E ES03T9

Red resid!e

$*)$3E4

1 !rine in DNA

o0idized . 3(forms allo0adial!ric acid)

1 Allo0an cond

form allo0ant

1 Ne!tralizatiom!re0ide

' f i 5M id


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eaction

mechanism9

'est for !rines5M!re0ide

h l 8 h ' t5' t f


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heeler18ohnson 'est5'est f.rimidines#R DNA 39DR*9A'+ S'ANDAR

: Clear, colorless Sol!tion '!rid,

so

& N/D

$ N/D

@ Clear sol!tion

hite reci-itate

9ellow

No r

< N/D

>";@P F 7' H?";L?G'E '7


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6 79

Clear olution%hite Preci)itate

Cleaar! Pur

: 79 Clea

%hite

1 79 7oPreci

' t f i idi 5h l 8


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+S$T$;E ES03T9

!r-le reci-itate

'est for .rimidines5heeler18o 'est

$*)$3E4

1 0idation of

-.rimidine to

Dial!ric Acid

1 Ne!tralizatio

Dial!ric Acid

TEST F+9 EA:E*T4


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TEST F+9

resence of -.rimidines

(C.tosine and 'h.mine)

EA:E*T4

%romine water

%a(3)&

'est for .rimidines5heeler 8o


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'est for .rimidines5heeler18o 'esteaction mechanism9


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C;7CL@;7

GDNA from white onion was isolated wherein the gro!-s ot $ =:


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$/=: gro!-s tested -ositive for deo0.riose in Dische

1 & o!t of > gro!-s tested -ositive for -hos-hate in 'est for

1 None of the gro!-s were ale to com-lete the M!re0ide a 8ohnson test


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R++R+NC+S

%o.er, R/ (&=:&)/ Biochemistry laboratory: Modern theory an


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techniques. New 8erse.4 earson +d!cation, Inc/

Ch!a1S!a, S/ (&=::)/ *aorator. man!al in %iochemistr./ U

hili--ines4 C2+ !lishing, Inc/

Clark, 8/, 8r/ (:>PH)/ !perimental Biochemistry / San rancisc

reeman and Com-an./

Clark, /, 2 Christo-her, / (&==:)/ An introd!ction to DNA4

S-ectro-hotometr., degradation, and the VrankengelV e0-erDNA handling and anal.sis/ Alerta, Canada4 Association for

*aorator. +d!cation/

Co!rtea!, 8/ (n/d/)/ Allium "epa. Retrieved from

htt-455eol/org5-ages5:=H@$


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N84 ile.1Interscience/

Smith, 8/#/ (&=:&)/ Principles of general# organic# $ biologica

New 9ork4 'he Mc#raw13ill Com-anies, Inc/

'ecan/ (&=:$)/ Nano7!ant -late4 A 7!ick g!ide/ A!stria4 'eca#m3/"oet, D/, "oet, 8/#/, 2 ratt, C// (&==H)/ Principles of biocheMassach!setts4 ile. 2 Sons, Inc/

alker, S/, 2 McMahon, D/ (&==H)/ Biochemistry demysti%edMc#raw13ill Com-anies, Inc/

experiment 8: isolation of dna from allium cepa

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