Magjet isolation with 0.25x volumes

Use this category for questions directly related to DNA manipulation (isolation, purification, sequencing, etc.) and questions regarding general PCR methodologies

Moderators: r.rosati, mchlbrmn

Magjet isolation with 0.25x volumes

Postby Diadumene » Oct 31 2016 10:09 pm

Hi-
I thought someone may be able to help me out. We have been using the Fisher Mag jet DNA isolation kits on tissues using 0.25x the 'recommended' volume (protocol attached). We get decent yields, but our lysate is quite viscous, causing pipetting issues. Because of this, we have decided to do the lysis step in the 'full' volume, and the remaining steps as 1/4 volumes. By changing the working volume mid-protocol, the digestion solution and the lysis buffer become our 'limiting' reagents, so we wanted to use lab-prepared solutions to augment the volumes. The digestion solution, I believe is just a basic lysis buffer with SDS, Tris-HCl, EDTA, and NaCl with Pro-K added. That's easy enough. But I don't know what Mag jet's version of lysis buffer is. I imagine there's some extra salt in it, but I don't know what else it contains. Does anyone have any idea or know how important that step is? I know they sell the buffers separately, but we're hoping to make them instead. Thanks.
Diadumene
newcomer
newcomer
 
Posts: 3
Joined: Oct 31 2016 9:58 pm

Re: Magjet isolation with 0.25x volumes

Postby relaxin » Nov 01 2016 6:45 pm

I am curious to know why you reduce the volume of buffer to 1/4 as suggested by the protocol? You may save the money by using less buffer, but you will reduce the efficiency of DNA isolation or induce the impurity of your DNA.

You may make your own buffers if you have the recipes. But do not try to use other recipes that are not given in the protocol. Them may not be compatible.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7190
Joined: Jan 11 2006 5:40 pm
Location: Mauna Kea

Re: Magjet isolation with 0.25x volumes

Postby Diadumene » Nov 01 2016 9:00 pm

Relaxin, the isolation works, and provides usable DNA for PCR. As you say, the reduction in buffer volume reduced efficiency-mostly regarding complete digestion, which is why we're looking to augment the volume. Lysis steps use fairly basic ingredients, so as long as our PCR works, there's no reason for us not to improvise. (These samples have only a single piece of tissue, no mixed communities or anything like that).
My basic question is what is the difference between the digestion solution and lysis buffer? Thanks.
Diadumene
newcomer
newcomer
 
Posts: 3
Joined: Oct 31 2016 9:58 pm

Re: Magjet isolation with 0.25x volumes

Postby relaxin » Nov 02 2016 4:18 pm

I am not familiar with your DNA isolation kit. In most DNA isolation protocols, the lysis and digestion buffers are actually one buffer. Some use SDS and proteinase K, while others use guanidine HCl and proteinase K. Proteinase K is active in either denaturing agent, which helps to lyse the tissue while proteinase K digests the proteins.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7190
Joined: Jan 11 2006 5:40 pm
Location: Mauna Kea

Re: Magjet isolation with 0.25x volumes

Postby Diadumene » Nov 07 2016 6:09 pm

Yes, I'm familiar with that. That's why I'm on this forum and asking. I also attached the protocol. Briefly, the tissue is incubated at 56 in the 'digestion' solution (which I would typically call a lysis buffer), which I presume is SDS, salt, lysozyme, pro K, EDTA, tris. After the overnight incubation, the 'lysis' buffer is added and the solution becomes quite cloudy. So I think there's more salt, but I'm not sure what else.
Diadumene
newcomer
newcomer
 
Posts: 3
Joined: Oct 31 2016 9:58 pm

Re: Magjet isolation with 0.25x volumes

Postby relaxin » Nov 08 2016 5:38 pm

I read the manual of the kit. They really do not tell you what are in the "digestion buffer" and "lysis buffer". The digestion buffer is for the enzyme to work. With the lysozyme the cells are lysed. So the "lysis buffer" may just contain some detergent, such as SDS. Since proteinase K is active in the presence of SDS, most people just use one buffer.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7190
Joined: Jan 11 2006 5:40 pm
Location: Mauna Kea


Return to DNA and General PCR Methods

Who is online

Users browsing this forum: Majestic-12 [Bot] and 6 guests

cron