The document provides three methods for extracting DNA from fungi. Method 1 uses lysis buffer, potassium acetate, phenol/chloroform/isoamyl alcohol, chloroform/isoamyl alcohol, NaCl, ethanol, PEG solution and TE buffer to isolate DNA. Method 2 uses extraction buffer, nuclei lysis buffer, sarkosyl, chloroform and sodium acetate to precipitate DNA overnight. Method 3 is a CTAB extraction method that uses extraction buffer, nuclei lysis buffer, sarkosyl, chloroform and ethanol wash steps to isolate fungal DNA.

1. DNA İZOLASYON YÖNTEMLERİ
1. CTAB METODU İLE DNA İZOLASYONU (BİTKİDEN)
MATERYAL
100 ml CTAB SOLÜSYONU İÇİN GEREKLİ MALZEMELER:
2 gr CTAB
10 ml 100mM TRİS HCl
4 ml 20mM EDTA
28 ml 1.4 M NACl
40 ml ddH₂O
1 gr PVP (polyvinylpyrrolidone)
% 0.2 mercaptoethanol (100 cc için 200 µl mercaptoethanol)
DNA İZOLASYONU İÇİN GEREKLİ OLAN DİĞER MALZEMELER:
izopropanol
Kloroform İzoamil alkol 24:1
%70’lik ethanol
TE tamponu
METOT 1
1 200 mg ezilmiş bitki yaprağı 2 ml’lik ependof tüpüne alınır
2) 900µl CTAB solüsyonu eklenir ve 65˚C de 1 saat sıcak su banyosunda bekletilir
3) Üzerine 900 µl 24:1 kloroform izoamil alkol eklenip 15 dk çalkalanır
4) 14000 rpm’de 15 dk santrifüj edilir
5) Süpernatant temiz bir ependof tüpü içerisine alınır ve üzerine 600 µl propanol eklenir ve bir gece
-20˚C’ de örnekler bekletilir
6)DNA’ları kaybetmeden solüsyon dökülür ve üzerine % 70 lik ethanolden 600µl eklenir ve 5-10 dk
çalkalamaya alınır
7) Ethanoller dökülür ve 3- 4 saat DNA’lar kurumaya alınır
8) 50-100µl ddH₂O veya TE tamponu eklenir, DNA çözülür ve -20˚C de DNA’lar saklanır.

2. METOT 2
1 200 mg ezilmiş bitki yaprağı 2 ml’lik ependof tüpüne alınır
2) 700µl CTAB solüsyonu eklenir ve 65˚C de 45 dk sıcak su banyosunda bekletilir
3) Üzerine 700 µl 24:1 kloroform izoamil alkol eklenir
4) 7000 rpm’de 5 dk santrifüj edilir
5) 600µl süpernatant temiz bir ependof tüpü içerisine alınır ve üzerine 360 µl propanol eklenir
6)14000 rpmde 5 dk santrifüj edilir ve süpernatatnt atılır
7) peletler % 70’lik ethanolle yıkanır
8) 14000 rpm’de 5 dk santrifüj edilir
9) süpernatantı dök peleti kurumaya bırak
10) 200µl TE tamponu eklenir, DNA çözülür ve -20˚C de DNA’lar saklanır.

3. 2. DNA Extraction Protocol (BİTKİ)
 Harvest fresh leaf tissue (1 to 2 g ) and snap freze in liquid nitrogen. Grind the tissue to a fine
powder in a chilled mortar with liquid nitrogen and glass particles (obtained by grinding a glass
Pasteur pipette) or acid washed sand (it is important to include glass particles or sand because of
the fibrous nature of the leaves )
 Add 7 ml of extraction buffer (preheated to 65 0
C ) to the powdered tissue in a 50 ml sterile screw
cup tube , mix well (without shaking ) , and incubate at 65 0
C for 30 min.
 Add an equal volume of chloroform/isoamyl alcohol (24:1) and mix gently by inversion .
 Centrifuge at 4.500 xg for 20 min. At room temperature. Transfer the supernatant into a sterile 30
ml Corex tube .
 Add one volume of cold isopropanol , mix gently by inverting the tube and incubate at – 200
C
for 30 min. to precipitate the DNA.
 Centrifuge at 12.00 x g for 20 min. at 4 0
C or hook DNA using sterile glass houk. Remove the
supernatant , wash the pellet with 75 % ethanol and leave to dry on the bench for 10 to 15 min.
 Dissolve the DNA pellet in mmmm1 ml of TE buffer. Add 1 ml of RNAase (10 mg/ml ) and incubate
at 37 0
C for 20 to 30 min.
 Add 2 ml of TE buffer, 1/10 th volume 3 M sodium acetate ,pH 8.0 and 2 volumes of % 95 cold
ethanol .Mix by inversion and incubate at -20 0
C for 30 min to precipitate DNA
 Centrifuge at 12.000 x g for 20 min. at 4 0
C or hook DNA out using sterile glass houk.Remove and
discard the supernatant ,wash the pellet with 75 % ethanol and leave to dry on the bench for 10-15
min.
 Dissolve in 1 ml of TE ,and store at 4 0
C .
Important !!!
Beware of chloroform and mercaptoethanol fumes as they are toxic.Discard the chloroform extract in the
container designed for it .
 Do not spin more than 5000 rpm ; CTAB ,can precipitate the DNA
c- Solutions required .
1 M Tris pH: 8.0
5 M NaCl
0.5 M EDTA pH: 8
3 M Sodium acetate pH: 5.2
10% CTAB.
10% sds
1X TE
Chlorophorm /isoamyl alcohol (24/1)
İsopropanol
Ethanol (1000 and 70 % )

4. Preparation of stock solutions
1M Tris pH 8.0 (500) dissolve 60.55 g Tris in 400 ml dH2O, adjust the pH to 8.0 with
conc HCl
Make up to 500 ml. autoclave.
5 M NaCl (500 mls) dissolve 146.1 g NaCl in 300 ml dH2O ,make up to 500 ml and
autoclave.
0.5 M EDTA (500 MLS) dissolve 93.05 g Na2EDTA.2 H2O in approx 100 mls H2O adjust
pH to
8.0 with NaOH pellets. Make up to 500 mls. Autoclave.
3 M Na acetate dissolve 40.82 g Na acet 3 H2O in 500 ml. adjust pH to 8.0 with
glacial
pH 8.0 (100mls) acetic acid. make up to 100 mls filter sterilise.
10 % sds (500 mls) dissolve 50 g SDS in 500 mls H2O
10 % CTAB dissolve at 10 g CTAB in 100 mls dH2O heat to dissolve
(100 mls)
1 X TE 50 mM Tris HCl ( pH8.0) to mM EDTA ( pH 8.0)
-Exraction procedure.
Extraction buffer Stock solutions Quant for 100 ml extr . buffer
3% (w/v)CTAB % 10 30 ml
1.4 M NaCl 5 M 28 ml
20 mM EDTA pH 8.0 0.5 M 4 ml
100 mM Tris HCl pH 8.0 1 M 10 ml
0.2 % (v/v) 2- Mercaptoethanol 200 ml
27.8 dH2O

5. FUNGAL DNA İZOLASYONU
METOD 1 (Alternaria spp)
Solutions:
1. Lysis Buffer
Final Cocentration 100ml 25ml Stock Solution
50 mM EDTA 10 ml 2.5 ml 0.5 M EDTA pH 8
100mM Tris 10ml 2.5ml lM Tris pH8
3% SDS 30 ml 7.5 ml 10% (w/v) SDS
Warm sterile distilled water 50 ml 12.5 ml
2. 8M Potassium Acetate
3. Phenol/Chloroform/Isoamyl Alcohol (25:24:1)
4. Chloroform/Isoamyl Alcohol (24:1)
5. RNase
6. 5 M NaCl
7. 100% Ethanol
8. 70% Ethanol
9. PEG Solution: 2.5 M NaCl, 20% PEG {8 ml 50% PEG, 10 ml 5M NaCl, 2 ml dH20)
10. TE (10 mM Tris pH 8 and 1 mM EDTA)
1. Grind lyophilized mycelium to line powder. Put amount to reach bottom of 100 µl mark on 1.5 ml centrifuge
tube.
2. Add 800 µl lysis buffer. Vortex each tube 5-10 sec. lncubate at 65 °C for 30-45 min. Centrifuge 15 min,
Transfer 700 µl of supematant to 1.5 ml tube.
3. Add 200 µl 8 M potassium acetate to each tube. Vortex briefly and place at -20 °C for 5-10 min or on ice
for 45 min-1 hour. The prep can also be stored at 4 °C until ready to resume. Centrifuge 15-30 min.
4. Transfer 700 µl supernatant to clear 1.5 ml tube. In fume hood, add 1 ml of Phenol/Chloroform/lsoamyl
Alcohol. Mix by inverting or gentle voitexing, centrifuge for 10 min.
5. Transfer about 600 µl aqueous phase to clear 1.5 ml tube, being careful to avoid taking up the interphase.
6. Add 500 pl of Chloroform/lsoamyl Alcohol Mix by inverting or careful vortexing. Centrifuge for 10 min.
7. Transfer 500 µl supernatant to new 1.5 ml tube.
8. Add 1/ 10th volume (50 µl) of 5 M NaCl and 2 volumes of 100% EtOH (1 ml), mix gently and put at -20C
for 1 hour or longer.
9. Spin down for 10 min and re-suspend completely in 500 µl dH2O
10. Add 350 µl PEG solution, mix well, and put at -20 °C for at least 1 hour or overnight at 4 °C
11. Spin down for l0 min. Discard supematant. Wash pellet with 500 µl of 70% EtOH twice. Air dry pellet for
several hours on bench or dry using Speedvac. Re-suspend pellet by adding 50 µl sterile TE or steril distilled
water. After sitting at room temperature or 4 °C for several min, pellets should resuspend completely through
gentle maniplation. Do not try to resuspend with a pipettor, as this will shear the DNA.
12. Add 1 µl of RNase (lmg/ml) (20 µg/ml iinal conc'n) and incubate at 37 °C for 2 hours.
13. Store at -20C. Load 2-4 µl on a test gel with 200ng lambda marker/Hindlll(4ul of 50ng/nl it marker/Hindlll)
Standard to estimate conc'n.

6. METOD 2
1. Frozen mycelia were placed in sterile 1.5 ml microcentrifugetubes,
2. 150 μl extraction buffer (0.35 M sorbitol, 0.1 M Tris, 0.005 M EDTA, pH 7.5,
0.02 M sodium bisulfite) was added, and tubes were vortexed.
3. Nuclei lysis buffer (150 μl) containing 0.2 M Tris, 0.05 M EDTA, pH 7.5, 2.0
M NaCl, and2% CTAB (hexadecyltrimethylammonium bromide) was added,
4. Followed by 60 μl of 5% sarkosyl (5 g N-lauryl sarcosine per 100 ml H2O),
and tubes were vortexed.
5. Then incubated at 65°C for 15 to 30 min.
6. One volume of chloroform:isoamyl alcohol (24:1) was added to each tube and
centrifuged for 15 min at 13,000 ´ g at room temperature.
7. The aqueous phase was removed to a new tube and the chloroform extraction
was repeated.
8. DNA was precipitated overnight at –20°C in 0.1 volumes of 3 M sodium
acetate, pH 8.0, and two volumes of cold 100% ethanol.
9. The supernatant was discarded, the pellets were washed with 70% ethanol, then
dried under vacuum centrifugation. DNA was resuspendedin TE (10 mM Tris-
HCl, 0.1 mMEDTA, pH 8.0).

7. METOD 3
CTAB Extraction of Fungal DNA
1. Grow mycelia in pea broth culture 7-10 days or until sufficient mycelia.
2. Harvest mycelia by vacuum filtration and freeze at -20 °C.
3. Add 150 µl Extraction Buffer, vortex. Grind mycelia with sterile Konte pestle.
4. Add 150 µl Nuclei Lysis Buffer and 60 µl 5% Sarkosyl, vortex to mix.
5. Incubate at 65 °C for 15-30 min.
6. Add 1 volume (~300µl) Chloroform (CHCl3): Isoamyl Alcohol (24:1), invert to mix.
7. Centrifuge 15 min, 12K rpm, room temperature.
8. Transfer aqueous phase to a new microfuge tube. Repeat chloroform extraction.
Centrifuge 15 min, 12K rpm, room temperature.
9. Transfer aqueous phase to a new tube. To aqueous phase add 0.1 volumes 3M
Sodium Acetate (NaOAc), pH 8.0 and 2 volumes of cold 100% Ethanol.
10. Allow DNA to precipitate overnight at -20 °C.
11. Centrifuge to pellet DNA, 10 min, 12K rpm, room temperature. Pour off supernatant.
12. Wash pellet twice with 70% Ethanol.
13. Dry pellet in speed vacuum.
14. Resuspend pellet in Te buffer, pH 8.0.
Extraction Buffer: 250 mlper LFormula (FW)
0.35 M Sorbitol 15.94 g 63.77 g C6H14O6 (182.2)
0.1 M Tris 3.03 g 12.11 g C4H11NO3 (121.1)
0.005 M EDTA (pH 7.5) 0.47 g 1.86 g C10H14N2O8Na2.2H2o (372)
0.02 M Sodium Bisulfite 0.95 g 3.8 g
Adjust pH to 7.5 with HCl. Do not autoclave and store at 4 °C.
CTAB (Nuclei Lysis Buffer): 250 mlper LFormula (FW)
0.2 M Tris 6.05 g 24.2 g C4H11NO3 (121.1)
0.05 M EDTA pH 7.5 4.65 g 18.6 g C10H14N2O8Na2.2H2o (372.2)
2.0 M NaCl 29.20 g 116.88 g NaCl (58.44)
2% CTAB 5 g 20 g C19H42NBr (364.5)
(cetyltrimethylammonium bromide)
5% Sarkosyl:
5 g N-lauryl sarcosine per 100 ml H2O. Autoclave.
3M Sodium Acetate:250 mlper LFormula (FW)
3M Sodium Acetate 61.52 g 246.09 g C2H3O2Na (82.03)
Adjust pH 8.0 with HCl and adjust volume to 1 liter. Dispense and autoclave. Store at
room temperature.
Buffers

8. 1M Tris HCl (pH 8.0): 100 mlper LFormula (FW)
1M Tris HCl 15.76 g 157.6 g C4H11NO3.HCl (157.6)
Add Tris into 80 ml H2O. Adjust pH with HCl. Bring to final 100 ml volume. Sterilize
by autoclaving.
0.5 M EDTA (pH 8.0): 100 mlper LFormula (FW)
0.5 M EDTA 18.6 g 186 g C10H14N2O8Na2.2H2O (372)
Add EDTA to 70 ml water and stir vigorously. Adjust pH 8.0 with NaOH. Adjust to
100 ml volume and autoclave.
Te Buffer (pH 8.0): 50 ml
10 mM Tris-HCl 0.5 ml of 1 M Tris-HCl (pH 8.0)
0.1 mM EDTA 0.01 ml of 0.5M EDTA
H2O 49.49 ml
Te Buffer (pH 8.0): 50 ml
10 mM Tris-HCl 0.5 ml of 1 M Tris-HCl (pH 8.0)
0.1 mM EDTA 0.01 ml of 0.5M EDTA
H2O 49.4 ml
70% Ethanol: 73 ml 95% ETOH + 27 ml H2O
METOD 4

9. DNA isolation protocol for A. rabiei
CTAB – buffer
2% (w/v) Cethyltrimethyl-ammoniumbromid
1.4 M NaCl
0.1 M Tris-HCl (pH 8.0)
20 mM EDTA
1. Harvest hyphae and spores directly from the Petri dish (see “A. rabiei – CMA growth
in Petri dishes”) by using a glass slide. Grind up the yield to a fine powder in mortal
with liquid nitrogen. Attention: since the fungal material is dense and wet when
harvested, one should pour the liquid nitrogen to the mortal, cool the mortal down,
and add the fungal material slowly, in small drops, to the liquid nitrogen.
Powdered tissue can be kept in the 50 ml tube at -20 °C for a few days.
2. Add powdered tissue to 50 ml tube containing (12 ml) preheated CTAB-buffer,
incubate for at least 30 min. at 60 °C while shaking.
3. Add 12 ml chloroform/isoamylalcohol [24:1 (v/v)] (USE THE HOOD!). Shake for 10
min. at approx. 60 rpm at room temperature.
4. Spin at 5000 rpm, 15 °C 20 min.
5. Transfer the upper aqueous phase to a new 50 ml tube, add 1 vol. isopropanol, mix
gently by inversion and incubate for 15 min. at room temperature.
6. Precipitate nucleic acids by centrifugation: 5000 rpm, 15 °C 20 min., discard the
supernatant and wash the pellet with 70% (v/v) ethanol. Centrifugation is optional.
7. Dry the pellet and resuspend it by adding 1000 µl TE (pH 8.0).
8. Store O/N at 4 °C or enhance suspension by incubating the tube at 60 °C for an
hour.
9. Add RNase A to final concentration of 10 µg/ml and mix gently, 1 h at 37 °C.
10. Add an equal vol. of chloroform/IAA and invert for at least 10 min., then centrifuge
for 15-20 min. at 2000-3000 rpm to separate phases. Remove upper phase into clean
2 ml tube.
11. Add an equal volume of isopropanol and incubate at room temperature for 15 min..
12. Spool or pellet the DNA (5000 rpm, 15 °C 20 min.), rince once with 70% ethanol and
allow to dry.
13. Re-suspend by adding 250-500 µl TE (pH 8.0).
14. For PCR purposes, dilute DNA with sterile double distilled water instead of TE.
Expected yield ranges 10-120 µl DNA
METOD 5
1.50 L ( ~100 samples)
300 ml (CTAB 10 %)
420 ml (NaCl 5 M)
150 ml (Tris 1 M)
60 ml (EDTA 0.5 M)
570 ml DDW

10. FUNGAL DNA MINIPREPARATION R.J. RODRIGUEZ
1) Grow fungus either as 100 ml culture or 4 plates with 8 ml liquid medium per plate
2) Collect mycelia on 2X cheesesloth and transfer to a weigh boat
3) Freeze-dry mycelia to completion (o/n)- can store dry mycelia at -20 C
4) Transfer mycelia to a mortar and add enough liquid nitrogen to cover mycelia.Let the liq.N2
evaporate and powder mycelia with a pestle.(can store powder in freezer).
5) Transfer powdered mycelia to falcon tubes (2006) to a level of approx. 2 ml,
6) Add enough lysis buffer to make a liqiud slurry (4-6 ml/sample) MUST BE FLUID.
7) Vortex samples 1-5 seconds.
8) Place samples at 65C for 20-30 minutes
9) Pellet debris at 10,000 rpm for 20 min. İn JA-20.1 rotor and transfer SN to a clean 15 ml polyallomer
tube.
10) Add 0.7 volumes of PEG/NaCl solution ,mix thouroughly ,and ice for 15-30 minutes .
11) Spin 5 minutes at 7000 rpm and resuspend in 2 ml TE buffer
12) After DNA has dissolved add 0.5 volume of NH4OAc,ice for 20 minutes ,and pellet protein ,RNA,REI
etc. At 10,000 rpm for 20 min.
13) Transfer S/N and add 0.6 volumes of Isopropyl alcohol (IPA) ,mix samples gently to spool out
DNA,and ice for 15-30 minutes.
14) Pellet DNA for 5 minutes at 5000 and resuspend DNA in 0.5 ml TE buffer.
15) Add NaCl to 0.1 M final and 2 volumes 95% EtOH ,gently mix and ice 15-30 minutes.
16) Pellet DNA for 5 minutes at 5000 rpm and resuspend DNA in 0.5 ml TE buffer .
17) Repeat NaCl /EtOH precipitation and resuspend DNA in 0.5 ml buffer.
18) For restriction analysis add spermidine to 4Mm in digestionreaction ; if DNA is not digesting, repeat
NaCl /EtOH and PEG/NaCl precipitations .Concentration of DNA (approx.200ng /ul ) should be such
that 30 ul is sufficient for a genomic digestion analyzed on a 20 x 20 cm gel .
Lysis Buffer FinalStock solution10 ml15 ml PEG/NaCl soln.
EDTA 150 mM 0.5 M 3 ml 4.5 ml 20 % PEG mw 8000
Tris 8.0 50 mM 1.0 M 0.5 ml 0.75 ml 2.5 M NaCl
Sarkosyl 2.0 % 20 % 1.0 m 15 ml
Protease 11 2 mg / ml 20 mg 30 mg
Water to volume
NH4OAc =Ammonium Acetate at 7.5 N =7.5 M
IPA =Isopropyl Alcohol
TE= 10 mM Tris 8.0 , 1Mm EDTA
JA-20.1 Rotor is a Beckmann rotor which holds 32 15 ml tubes
EtOH =Ethanol
STOK SOLÜSYONLAR

11. 1) 1 M Tris –HCI pH:8
Tris Base: 121,1 gr/ mol x 1 M =121,1 gr /lt = 60. 550 gr /500 cc PH, HCL ile ayarlanır.
2) 5 M NaCl mw=58.44 /87.66
5 x 58.44 = 292.20 gr /lt = 146.10 gr /500 cc
3) 0.5 M EDTA pH :8
0.5 M X 372.2 =186.1 gr/lt = 93.5 gr/ 500 ml PH NaOH ile ayarlanır.
4) TE Buffer pH:8
10 Mm Tris Base= 0.01 x121.1
=1.211 gr /lt
= 0.6055 gr/500 cc
1 mM EDTA =0.0001X372.2
=0.3722 gr/lt
= 0.1861 gr/500 cc
TE Buffer could be prepared from stock 1 and 3 .
1 M → 1000 mM 10 M→100 mM
10 cc 1 M Tris HCL
990 cc water→ total 1000 cc
0.5 M →500 mM EDTA
pH 8 EDTA 0.5 ml to 250 ml Tris HCl 2.5 ml to 250
5) 5 M KAoC ( Potasyum asetat)
5 x98.14 = 490.70 gr/lt
= 122.675 gr / 250 cc
6) 20 % SDS ( No autocleve sterilization)
SDS solved 65 0
C
7) 3 M NaAoc ( Sodyum asetat) pH:4.8
3 x 136.08 = 408.24 gr/ lt

12. =102.6 gr/250 cc
NaAoC stock solution ; pH is adjusted with glasiyal acetic acid
8) Buffer ‘’S’’ (250 ml )
a) 100 mM Tris HCl pH:8
b) 50 mM EDTA pH: 8
c) 500 mM NaCl
d) 10 mM Mercaptoethanol
e) 2% SDS
100 mM Tris HCl
1 M Tris HCl = 1 ‘ e 10 oranında sulandırılacak
X 250
X= 25 cc 1 M Tris HCl
50 mM EDTA
0.5 M EDTA = 500 mM 1 ‘ e 10 oranında sulandırılacak
50
250 cc için 25 cc 0.5 M EDTA, 225 cc water ,
500 mM NaCl için ;
5 M NaCl = 10 mM NaCl
= 25 cc 5 M NaCl 225 cc water
% 2 SDS ; 20 % SDS
100 20
X 2 X= 10 cc / 100 cc için
250 cc için ; 25 cc 20 % SDS
9) % 70 + % 100 EtOH (100 ml )
10) Iso –propanol
11) SDOW
12) Glicerol
13) Eppf tüp
14) Eips
Preperation of Mercaptoethonal

13. 10 mM = 0.01 M = 0.07813 gr/ lt
Looding dye
% 40 sucrose
50 mM EDTA
50 mM Tris
0.25 % Bromophenol Blue
Spermidine ; dxsolve TE
100 mM 127.4 mg/ 5 ml
100 mM spermidine
254,6 X 100
=25.46 gr / lt
1000
25.46 x 1000 ML
X 10 ml X = 254.6 / 10 cc
Looding Dye
 Ficoll % 10 4 ml (0.5 M stock )
 Na2EDTA 0.1 M 2 ml ( % 20 stock )
 SDS % 2 up to 20 ml with x1 TE Add some Bromophenol blue
 TE X1
ETD Bromide ;
2 lt water
400 ml EB (10 mg/ml ….)
Looding dye
 40 % glycemol
 2 mM EDTA

14.  …. Bromophenol blue
+
1 kb ledder … 1 ml of looding dye
Preperation of RNAase
a) 10 mg RNAase in tube
b) Add 10 ml of 1 M Tris-HCl Stock
c) Add 30 ml of 5 M NaCl –stock
d) Add 960 ml H2O
↘ seperate ↙
500 ml 500 ml
↓
Boil for 15 ml
↓
Leave at room temperature ( RT) for 30 min.
↓
Keep -20 0
C
Preperatin of ‘’ proteinese K ‘’
Prepore stock solution
↓
Solve 100 mg Proteinese K in 4 ml TE buffer
↓
100 mg /4 ml
4000 ml 100000 Mg
X 100 mg
X= 4 ml
↓ 4ml contains 100 mg Pro. K. 40 use 4x15 = 60 ml for 15 ml DNA preperation
Keep -20 0
C
Mercaphtoethanol ;add it into ‘’ Buffer S ’’ after autocleve. (10 mM mercaphtoethonol ) for 250 cc ‘’Buffer
S’’
1000 mM → 78.13
10 mM → 0.7813 /lt

15. 250 ml için → 0.195 gr
Yoğunluk ↚ 1.12 gr /ml 1000 ml
0 .195 gr X
X= 175 ml mercaphtoethonol for 250 cc buffer ‘’ S’’
X= 2.34 x 100 =2092. 768 mlpex for 30 mM mercaphtoethonol
1.12
1 liter of 10X TBE
Chemical Amount
Tris base 242 g
boric acid 165 g
EDTA (solid) 24.6 g
Dilute to 3 liters and stir till all is dissolved. Note: TBE will precipitate over time. Suggestions to
reduce this include using very clean glassware or making up 5x stock.
To make 2 liters of 1x running buffer, bring 200 ml of 10X TBE up to 2000 ml with distilled water.
1 liter of 50X TAE
Chemical Amount
Tris base 242 g
glacial acetic acid 57.1 ml
0.5 M EDTA 100 ml
Bring solution up to 1 liter with distilled water.
To make 2 liters of 1x running buffer, bring 40 ml of 50X TAE up to 2000 ml with distilled water.