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Manual DNAExtraction

This document provides instructions for extracting and purifying DNA from ancient dental pulp samples using a silica spin column method. Key steps include: 1) Preparing lysis buffer and binding buffer solutions and resuspending dental pulp samples in lysis buffer overnight. 2) Clearing supernatant containing DNA into binding buffer and passing it through a silica spin column to bind DNA. 3) Washing and eluting DNA from the column using pre-warmed water and collecting the purified DNA. 4) Quantifying the purified DNA using Qubit and performing qPCR to test DNA concentration.

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souha guebebia
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© © All Rights Reserved
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Download as PDF, TXT or read online on Scribd
46 views

Manual DNAExtraction

This document provides instructions for extracting and purifying DNA from ancient dental pulp samples using a silica spin column method. Key steps include: 1) Preparing lysis buffer and binding buffer solutions and resuspending dental pulp samples in lysis buffer overnight. 2) Clearing supernatant containing DNA into binding buffer and passing it through a silica spin column to bind DNA. 3) Washing and eluting DNA from the column using pre-warmed water and collecting the purified DNA. 4) Quantifying the purified DNA using Qubit and performing qPCR to test DNA concentration.

Uploaded by

souha guebebia
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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DNA extraction and purification from ancient dental pulp

- Prepare 1 mL of Lysis buffer (0.45 M EDTA pH 8.0, and 0.25 mg/mL proteinase K in nuclease-
free water) per ~100 mg of stool sample
o For 500uL

▪ 45uL nuclease free water


▪ 450uL of 0.5M EDTA
▪ 5uL of 25mg/ml proteinase K

- Prepare 5mL of 5% Tween-20 (4.75mL nuclease-free water + 250uL Tween-20) The Tween-20 is
very viscous -> cut the tip before pipetting, homogenize on a rotating wheel
- For each sample, prepare 5mL of binding buffer (5 M guanidine hydrochloride, 40% (vol/vol)
isopropanol, 0.05% Tween-20 and 90 mM sodium acetate (pH 5.2)).
o For 50mL (pour 5mL de binding par échantillons)

▪ 23,88 g of guanidine hydrochloride (We can proceed without in the case you can
get it)
▪ 20mL isopropanol
▪ 500ul of freshly prepared 0.05% Tween-20
▪ 1,5 mL sodium acetate (pH 5.2)
▪ add water up to 50mL

- Check that pH is under 7.5 for lysis and binding buffer


- Add 1ul of TISS (Extern DNA= control) in each sample (beforehand resuspended in 990ul of
nuclease free water)
- Resuspend in 500uL of Lysis Buffer and vortex
- Incubate between 12-18h at 37°C using a thermomixer (500rpm) or on a rotative wheel
(recommended).
- The next day, pellet the remaining stool residues by centrifuging for 2min at 16,000g (or rcf)
- Transfer the clear supernatant into a fresh and DNA free 50mL (if possible LoBind) falcon tube
- Add 5mL of binding buffer and vortex

For the steps below you can use any spin column and follow the manufacturer steps. The final
goal is to elute DNA using spin column.

- Transfer 600uL of sample on a Qiagen MinElute Silica Spin column and centrifuge for 30sc at
16000g (You can use Qiagen fast prep or any spin column from any manufacturer).
- Trash the flow-through and repeat the previous step until the full 6mL of sample have been
adsorbed on the column
- Discard the flow-through and centrifuge for 1 extra minute
- To wash, add 600µL Buffer PE to the MinElute column and centrifuge for 1 min at 16000 g (see
step 6 of the manufacturer’s protocol
https://www.qiagen.com/at/resources/download.aspx?id=fa2ed17d-a5e8-4843-80c1-
3d0ea6c2287d&lang=en)
- Pre-warm 100 uL of nuclease-free water (or steril water) at 37°C
- Trash the flow-through and centrifuge again for 2min at 16,000g in order to remove any eventual
traces of wash buffer
- Trash the collection tube and place the column into a fresh 1.5mL LoBind Eppendorf tube
- To elute the DNA, add 12.5ul of pre-warmed nuclease-free water directly on the membrane of the
column
- Stand the column for 3 min at R.T
- Centrifuge for 1min at 16,000g
- Eventually repeat the elution steps using 12.5uL of pre-warmed water (this extra step might
improve elution efficiency, final volume 25uL)
- The eluate contains the purified DNA
- Perform Qubit quantification (HS)
- 5 qPCR are performed from one sample as follow: 1 with 5 ul of non-diluted DNA ; with 5 ul of
1/25 diluted DNA ; 5ul of 1/50 diluted DNA ; 5ul of 1/100 diluted DNA ; 1ul of pure DNA targeting
TISS sequence.

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