Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > General Science Forums > Biochemistry Forum > DNA Forum
Register Search Today's Posts Mark Forums Read

DNA Forum Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.


DNA storage at -80

DNA storage at -80 - DNA Forum

DNA storage at -80 - Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 07-25-2011, 05:25 PM
Pipette Filler
Points: 295, Level: 5 Points: 295, Level: 5 Points: 295, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2011
Posts: 19
Thanks: 0
Thanked 2 Times in 2 Posts
Default DNA storage at -80



I have isolated DNA from buffy coat, roughly 30 microliters and will be storing at -80. I would like to add Tris-EDTA to protect during long time storage. I was thinking of making the total volume 200 microliters for storage but I'm unsure as to wether this is satisfactory. Likewise whether TE is suitable for storage at -80. Thoughts and suggestions would be great.

Cheers,

-M
Reply With Quote
  #2  
Old 07-27-2011, 12:35 PM
Pipette Filler
Points: 768, Level: 15 Points: 768, Level: 15 Points: 768, Level: 15
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2010
Posts: 2
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: DNA storage at -80

Tris is generally not a good buffer for low temperatures because it has significant dependence of pH on temperature. See Sieracki, N.A., et al. Chem. Commun., 2008, 823825. This may be OK for just plain DNA if you start with pH 8.5 at room temperature.

You can easily store 200 microliters, just do not use snap cap tubes. The screw cap tubes are much better in preventing evaporation, so your solution will not evaporate and change the DNA concentration.

Richard Owczarzy
Reply With Quote
The Following User Says Thank You to Richard_Owczarzy For This Useful Post:
admin (08-11-2011)
  #3  
Old 07-27-2011, 02:43 PM
luisillo's Avatar
M.D/Ph.D
Points: 4,061, Level: 42 Points: 4,061, Level: 42 Points: 4,061, Level: 42
Activity: 25% Activity: 25% Activity: 25%
 
Join Date: Jul 2010
Location: Mexico
Posts: 353
Thanks: 19
Thanked 98 Times in 90 Posts
Default Re: DNA storage at -80

Tris-EDTA is the choice to store samples in the refrigerator (4-8C), but If your planning to freeze your samples you better dilute them in water.

Another thing you should do is to store the necessary aliquots of the samples in order to avoid repeadly freeze-thaw cycles as this may damage DNA quality. This way, when you need DNA you can thaw an aliquot tube and you'll have more of that DNA in the freezer if you needed.

Good luck
Reply With Quote
The Following User Says Thank You to luisillo For This Useful Post:
admin (08-11-2011)
  #4  
Old 07-28-2011, 07:22 PM
Pipette Filler
Points: 295, Level: 5 Points: 295, Level: 5 Points: 295, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2011
Posts: 19
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: DNA storage at -80

I will actually be storing DNA and RNA at -80. What would be a good ratio of water to add to each sample for storage at -80?
Reply With Quote
The Following User Says Thank You to moleculargeek21 For This Useful Post:
admin (08-11-2011)
  #5  
Old 07-29-2011, 03:37 PM
luisillo's Avatar
M.D/Ph.D
Points: 4,061, Level: 42 Points: 4,061, Level: 42 Points: 4,061, Level: 42
Activity: 25% Activity: 25% Activity: 25%
 
Join Date: Jul 2010
Location: Mexico
Posts: 353
Thanks: 19
Thanked 98 Times in 90 Posts
Default Re: DNA storage at -80

It depends on your further assays with them. For RNA, store as much as the RT protocol calls for, and for DNA aliquots of 10-20 ul should be fine, then again is up to you to decide which depending how much you'll need for your further assays.
Reply With Quote
The Following User Says Thank You to luisillo For This Useful Post:
admin (08-11-2011)
  #6  
Old 08-11-2011, 01:17 PM
Pipette Filler
Points: 295, Level: 5 Points: 295, Level: 5 Points: 295, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2011
Posts: 19
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: DNA storage at -80

I will also be isolating buffy coat and storing at -80, I was just going to store the pelleted/washed buffy coat in cryo-vials and store directly in the freezers without a buffer. Not sure if this is recommended
Reply With Quote
  #7  
Old 08-11-2011, 03:16 PM
Pipette Filler
Points: 295, Level: 5 Points: 295, Level: 5 Points: 295, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2011
Posts: 19
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: DNA storage at -80

What considerations might I take for storing buffy coat at -80
Reply With Quote
  #8  
Old 08-12-2011, 05:49 PM
Warthaug's Avatar
Post-Doc
Points: 2,383, Level: 31 Points: 2,383, Level: 31 Points: 2,383, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Location: London, Canada
Posts: 203
Thanks: 1
Thanked 68 Times in 61 Posts
Default Re: DNA storage at -80

You'll need to put the buffy coat in 20% DMSO. Otherwise, ice crystals will shear your cells open, releasing all kinds of stuff that may damage your DNA. DMSO will prevent ice crystal formation, thus keeping stuff where it should be.

Simply add 20% DMSO (% by volume) to your sample and freeze. Ideally you'll want to freeze using an isopropanol cell freezer, as this maximizes the effectiveness of the DMSO. By cell freezer, I mean a Mr Frosty:
[Only registered users see links. ]

Bryan
Reply With Quote
  #9  
Old 08-18-2011, 08:40 PM
Pipette Filler
Points: 295, Level: 5 Points: 295, Level: 5 Points: 295, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2011
Posts: 19
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: DNA storage at -80

would you dilute the stock DMSO with DI water or Nuclease free water?
Reply With Quote
Reply

Tags
-80 , dna , storage , tris edta


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Cell Storage aftabac Cell Biology and Cell Culture 0 10-25-2010 08:23 PM
RNA Storage : Short and Long-term Storage bioinfofreak Molecular Biology Articles and Protocols 10 09-13-2010 04:10 AM
Storage of Purified Proteins danfive Protein Science 4 07-01-2009 02:14 AM
Long Term Storage of SDS-PAGE Gels dnamolecule SDS-PAGE Gel Electrophoresis Forum 1 08-28-2008 06:34 AM
Microarray slide storage Charles Harris Protocols and Methods Forum 0 04-29-2007 05:13 PM


All times are GMT. The time now is 12:03 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.16108 seconds with 16 queries