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Buffer composition for miniprep kit

miniprep

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6 replies to this topic

#1 sorcener

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Posted 21 May 2012 - 10:04 AM

Hi every one!

I'm using the invitrogen PureLink quick plasmid miniprep kit. I read somewhere else here you can re-use the comumns and as im run off of them i would like to try.

But I'm runnung out of buffers too. The kit have this buffers:

R3 (resuspension) which cotains tris HCl 50mM and EDTA 10mM
L7 (lysis): NaOH 200 mM 1%SDS

But the rest it does not say the compositin, I can guess accoding to other protocols that they must have some salts and alcohol, but do someone knows the exact composition of buffers:

N4 (precipitation buffer)
W9 and W10 (wash buffers)

I would really appreciate your help

#2 bob1

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Posted 21 May 2012 - 12:44 PM

In general minipreps and the larger versions have 3 major solutions: The resuspension buffer is usually tris (25 mM, pH 8), EDTA (10 mM) and glucose (50 mM). The lysis buffer is always NaOH (0.2 N) and SDS (1%), and the neutralization buffer is always an acetate (usually 3 M NaoAC pH to 4.8 with glacial acetic acid). The wash buffers will be 70% ethanol and/or 100% Isopropanol.

What is exactly in your kit is commercially sensitive information, so you won't get much help from Invitrogen... you can do these without columns you know?

#3 phage434

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Posted 21 May 2012 - 01:37 PM

The neutralization buffer is usually potassium acetate, as this will precipitate the dodecyl sulfate from SDS as a white precipitate, which helps to trap the genomic DNA during a spin down.

For column binding, the neutralization buffer also has a chaotropic compound such as guanididium hydrochloride. See the Qiagen buffer compositions here:
http://openwetware.o.../Qiagen_Buffers

Bob1 is correct. After neutralization with KOAc and spin down, the DNA can be directly precipitated from the supernatent, washed with 70% ethanol, and used.

#4 sorcener

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Posted 22 May 2012 - 05:59 PM

thanks fo the advise. Yes I know I can do it without the column, but i have been said homemade minis are not so clean, or do i have to use phenol/chlorophorm.

#5 bob1

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Posted 23 May 2012 - 12:56 PM

I get very clean DNA out of home made minipreps, I use a PEG precipitation procedure from the ABI sequencing guide.

#6 memari

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Posted 24 May 2012 - 03:38 PM

http://www.genetics....ls_Miniprep.htm

This Plasmid Purification is based on the small size and supercoiled nature of plasmid DNA.
EDTA causes instability in Membrane.
SDS solubilises the proteins and lipids of membrane which ends to disrupture in membrane of bacteria.
When you change the pH of solution with adding NaOH to 12-12.5 pH, Hydrogen Bands in Chromosomal DNA break and two strands of DNA are seperated.
But DNA of Plasmid stays intact because of the supercoiled nature of it.
After adding Acetat, Plasmid stays soluble but Chromosomal DNA precipites.
Plasmid precipites after adding IsoPropanol.
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Babak Memari
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Babak Memari

#7 zorrooo

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Posted 17 December 2013 - 07:57 AM

The most difficult buffer for miniprep is the neutralization buffer (columns).

 3 M NaoAC pH to 4.8 with glacial acetic acid as stated above is ok for subsequent ethanol precipitation but not for spin columns as DNA does not bind withouth at least 25% alcohol (ethanol or isopropanol).

Adding ethanol 25-30% to 3 M NaoAC pH to 4.8 should be ok.

The denaturant Guanidinium-HCl could be theoretically replaced by ethanol 70% by washing (twice to enhance the effect of denaturation) with wash buffer (10 mM Tris-HCl pH 7.5, 80% ethanol (wash buffer has ethanol which is also a denaturant)(http://en.wikipedia....haotropic_agent)).

We used a similar approach with good results.

I would try to avoid completely Guanidinium-HCl as this is a bit nasty eventually.







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