Protocol of DNA isolation using Phenol: Chloroform method isoamyl alcohol

method
In addition to this we also learned the phenol chloroform method details of which are as
followed1.

First of all sample is chopped with knife or blade into small pieces than kept into
eppondrof tube, meat sample weight up to 80-100 mg.

2. Add 500 l lysis buffer to it.

3. Add 50 l 20% SDS.
4. Add 20 l of proteinase K to each eppendorf. ( Stock 20 mg/ml)
5. Keep at 56 0C on Hybridization oven or Rocking Water bath overnight.
6. Add equal volume of saturated phenol to all samples except hair samples and shake
properly for 10 min. by hand and then centrifuge for 10 min at 10,000 rpm and 10 0C.
7. Take the supernatant and add it to 300 l of chloroform: isoamyl alcohol (24:1) and 300 l of
phenol and again centrifuge for 10 min at 10 0C and 10,000 rpm.
8. Take supernatant and add 600 l of chloroform: Isoamyl alcohol (24:1).
9. Take the supernatant very carefully and 0.1 volume of 3M Sodium acetate ( pH 5.2).
10. Then add 0.6 volume (60%) of isopropyl alcohol or 2 volume ethanol to it.
11. Keep the mixture at 4 0C to settle down the DNA for 1 hrs.

12. Again centrifuge at 10 0C or 10 min and 10,000 rpm.

13. Discard the supernatant and add 500 l of 70% ethanol to it and centrifuge again at 10 0C for
10 min and 10,000 rpm.
14. Discard the supernatant carefully and add 100% ethanol to the pallets and mix gently and
again centrifuge.
15. Discard the supernatant and keep the eppendorf on block heater for 10 min to dry the
extracted DNA.
16. Dissolve the DNA in Tris EDTA buffer ( amount according to size of pallet).
17. Keep at block heater again to dissolve the DNA.
Extraction of DNA using DNeasy Tissue QIAGEN Kit:1. Cut up to 25 mg tissue into small pieces, place in a 1.5 ml micro centrifuge tube , and add 200
l buffer ATL.
2. Add 20 l Proteinase K, mix by vortexing, and incubate at 55 0C until the tissue is completely
lysed. Vortex occasionally during incubation to disperse the sample, or place in a shaking
water bath or on a rocking platform.
3. Vortex for 15 sec. Add l buffer AL to the sample, mix thoroughly by vortexing. Then add 200
l (96-100%) ethanol to it. Mix again thoroughly.

4. Pipet the mixture into a DNeasy Mini spin column in a 2 ml collection tube. Centrifuge at
8000 rpm for 1 min. Discard flow through and collection tube.
5. Place the spin column in new 2 ml collection tube. Add 500 l buffer AW1. Centrifuge for 1
min. at 8000 rpm. Discard flow through and collection tube.
6. Place the spin column in a new 2 ml collection tube. Add 500 micro liter buffer AW2.
Centrifuge for 3 min at 14000 rpm. Discard flow through and collection tube.
7. Transfer the spin column to a new 1.5 ml or 2 ml micro centrifuge tube and add 200 l buffer
AE for elution. Incubate for 1 min at room temperature. Centrifuge for 1 min at 8000 rpm.

DNA Extraction from Fecal Pellets by Qiagen kit.

1. Weighed 180220 mg fecal pellets in a 2 ml microcentrifuge tube and placed tube on ice.
2. Added 1.6 ml Buffer ASL to each stool sample. Vortexed continuously for 1 min or until
the fecal sample is thoroughly homogenized.
3. Centrifuged sample at full speed for 1 min to pellet fecal particles.
4. Pipetted 1.4 ml of the supernatant into a new 2 ml microcentrifuge tube and discarded the
pellet.
5. Added 1 InhibitEX Tablet to each sample and vortexed immediately and continuously for
1 min or until the tablet is completely suspended. Incubated suspension for 1 min at room
temperature to allow inhibitors to adsorb to the InhibitEX matrix.
6. Centrifuged sample at full speed for 3 min to pellet fecal particles and inhibitors bound to
InhibitEX matrix.

7. Immediately after the centrifuge stops, pipetted all of the supernatant into a new 1.5 ml
microcentrifuge tube and discarded the pellet. Centrifuged the sample at full speed for 3
min.
8. Pipetted 25 l proteinase K into a new 2 ml microcentrifuge tube.
9. Pipetted 600 l supernatant from step 7 to the 2 ml microcentrifuge tube containing
proteinase K.

Fig 5- DNA extraction by column based Qiagen kit.

10. Added 600 l Buffer AL and vortexed for 15 s and Incubate at 70 C for 10 min.
11. Added 600 l of ethanol (96100%) to the lysate, and mixed by vortexing.
12. Labeled the lid of a new QIAamp spin column provided in a 2 ml collection tube.
13. Carefully applied 600 l lysate from step 12 to the QIAamp spin column without
moistening the rim. Closed the cap and centrifuged at full speed for 1 min. Placed the
QIAamp spin column in a new 2 ml collection tube, and discarded the tube containing the
filtrate.
14. Carefully opened the QIAamp spin column, applied a second aliquot of 600 l lysate and
centrifuged at full speed for 1 min. Placed the QIAamp spin column in a new 2 ml
collection tube, and discarded the tube containing the filtrate.
15. Repeated step 14 to load the third aliquot of the lysate onto the spin column.
16. Carefully opened the QIAamp spin column and added 500 l Buffer AW1.
17. Closed the cap and centrifuged at full speed for 1 min. Placed the QIAamp spin column
in a new 2 ml collection tube, and discarded the collection tube containing the filtrate.
18. Carefully opened the QIAamp spin column and added 500 l Buffer AW2. Closed the cap
and centrifuged at full speed for 3 min. Discarded the collection tube containing the
filtrate.
19. Transferred the QIAamp spin column into a new, labeled 1.5 ml microcentrifuge tube.
Carefully opened the QIAamp spin column and pipetted 200 l Buffer AE directly onto
the QIAamp membrane. Closed the cap and incubated for 1 min at room temperature,
then centrifuged at full speed for 1 min to elute DNA.
20. For long-term storage, kept the eluted at 20 C.

Quality assessment of isolated DNA using agarose gel electrophoresis

1. Prepared the casting tray for casting the agarose gel by making it grease free using 70%
ethanol.
2. Prepared 2% agarose in TAE buffer (50 ml) (0.8 g in 100 ml TAE buffer). Boiled to
dissolve in microwave oven
3. Prepared 2% agarose in TAE buffer (50 ml) (0.8 g in 100 ml TAE buffer). Boiled to
dissolve in microwave oven.
4. Added Ethidium Bromide (EtBr) (2 l/100 ml.) when gel temperature reaches about 50 oC
and swirled taking care that there is no air bubble formation.
5. Placed the comb in the casting tray and poured the prepared gel without formation of air
bubbles into it and allowed for polymerization.
6. The sample to be loaded was mixed with 5 X gel loading dye.
7. The comb was removed carefully to avoid deformation of wells.
8. The casting tray was now placed in the electrophoresis tank and 0.5 X TBE buffer was
poured into it.
9. The prepared samples were then loaded into the wells and the gel was electrophoresed at
100 volts for 30-60 mins.
10. Visualized the gel under the UV Transilluminator when the dye reaches 3/4th the length
of the gel.

Quantity

Assessment

of

isolated

DNA

using

GeneQuant

Pro

Spectrophotometer (Amersham Biosciences)

1. Switched on the instrument and waited for few minutes so that could read and locate Ref
1 and Ref 2 and calibrate itself.
2. As display panel shows Instrument Ready, selected and pressed DNA option on control
panel and inserted reference sample in cuvett (500 l TE) into the respective block and
pressed setup then instrument reads TE as blank.
3. Hereafter, set TE as blank, display panel shows Insert DNA Sample then added 1.0 l of
DNA sample in 500 l TE. Mixed thoroughly and placed the cuvett in to the respective
block.
4. Pressed Enter on control panel and instrument showed final DNA concentration on the
screen.
5. Noted down the values carefully and cleaned the cuvett and instrument after use.

Polymerase Chain Reaction

To amplify specific gene from genomic DNA, PCR reaction were set as follows. The
reaction mixture for PCR consisted of Genomic DNA

25ng (1 l)

Forward Primer

0.2l (2 pmol)

Reverse Primer

0.2l (2pmol)

10 X Buffer

1 l (1X)

Magnesium chloride

0.6 l

BSA

1 l

dNTPs (2.5 mM)

0.2l (100M)

Taq polymerase(3U/l)

0.2l (0. 2units)

Double distilled water to final volume of

5.6 l

Preparation of Hot star master mix

Hot star master mix

5 l

Forward Primer

0.2l (2 pmol)

Reverse Primer

0.2l (2 pmol)

Double distilled water to final volume of

5.6 l

Primer sequences used to amplify cytochrome b, 16S, and D-loop

Gene

Primer sequence
F

5-GCCCCTCAGAATGATATATTTG-3

5-CCATCCAACATCTCAGCATGATGAAA-3

5-TACCATGAGGACAAATATCATTCTG-3

Cyt b

R
F

5-CCTCCTAGTTTGTTAGGGATTGATCG-3
5'-CCCAAAGCTGAAATTCTACTTAAACTA-3

D-loop

5'-GCATGGGGCATATAATATAATGTAC-3

16s

Procedure1.
2.
3.
4.

Prepare master mix.

Add 9l master mix to all of the labeled PCR tube.
Add 1l of template DNA to each tube.
Mix the contents by short spin of PCR tubes.

5. Keep the tubes in thermal cycler.

6. Run PCR under desirable conditions.
The PCR reaction was typically programmed for the following thermal regimens for the
following genes
1. Cytochrome b Gene
Initial Denaturation

94C for 5 minutes

Denaturation

94 C for 45 seconds

Annealing

55 C for 30 seconds

Extension

72 C for 30 seconds

40 cycles

Final extension at 72 C for 10 minutes and stored at 4 C or -20 C.

Condition for Hot star master mixInitial Denaturation

95C for 15 minutes

Denaturation

95 C for 40 seconds

Annealing

55 C for 45 seconds

Extension

72 C for 1:15 seconds

40 cycles

Final extension at 72 C for 15 minutes and stored at 4 C or -20 C.

2. 16 S Gene
Initial Denaturation

94C for 5 minutes

Denaturation

94 C for 45 seconds

Annealing

55C for30 seconds

Extension

72 C for 30 seconds

40 cycles

Final extension at 72 C for 10 minutes and stored at 4 C or -20 C.

3. D-loop Gene

Initial Denaturation

94C for 5 minutes

Denaturation

94 C for 45 seconds

Annealing

54C for 30 seconds

Extension

72 C for 30 seconds

40 cycles

Final extension at 72 C for 10 minutes and stored at 4 C or -20 C.

Determination of quality and quantity of PCR productPCR product was run on 2% agrose gel.

Exo Sap treatment

1. Approx 1 l -1.5 l PCR products are taken in PCR plate.
2. Add 0.5 l Exo-Sap-I.

Condition for the activation of Exo SapI

1. 37 C for 20 minutes
2. 80 C for 15 minutes

CYCLE SEQUENCING
Thermostable DNA polymerase allows the sequencing reaction to be cycled through
alternating periods of thermal denaturation, primer annealing, and extension to increase the
single levels generated from template DNA. This amplification process employs a single
primer, so the amount of product increases linearly with a number of cycles. A cycling
protocol is especially useful when the amount of template is limiting is limiting or the
sensitivity of the detection system is low.

Cycle sequencing PCR

Preparation of master mix

COMPONENTS

PER REACTION

RR Big dye TM mix

5X sequencing buffer
Primer
PCR product
Sterile water
Total Volume

0.15l
0.8l
0.2l
1.0l
4.0l
9.0l

PCR condition for cycle sequencingInitial Denaturation

96C for 1 minute

Denaturation

96 C for 0.10 seconds

Annealing

50C for 0.05 seconds

Extension

60 C for 4 minutes

30 cycles

Stored at 4 C or -20 C

Sequencing Reaction Clean up

1. Added 40 l (24:1 alcohol: sodium acetate) in each wells of PCR plate.
2. Incubated at room temperature for 15 minutes
3. Centrifuged at 4000 rpm for 20 minutes
4. Decant it carefully
5. Added 100 l 70% ethanol in each wells
6. Centrifuged at 4000 rpm for 20 minutes
7. Again decant it.
8. Dry it for 5 minutes
9. Added 8 l Hidi (formamide) in each well.
10. Denature at 95C for 3 minutes
Load in applied Biosystems 3130 Genetic Analyzer for sequencing.