Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Cleaning up DNA for microinjection

Cleaning up DNA for microinjection - Protocols and Methods Forum

Cleaning up DNA for microinjection - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 11-28-2006, 06:25 PM
Tom Anderson
Guest
 
Posts: n/a
Default Cleaning up DNA for microinjection



Hi everyone,

Back with another purification question. I'm trying to do some DNA
microinjection into cells, but am finding that i'm not getting flow
through my needles. When i use some control DNA from a colleague, it works
fine, so it seems my DNA solution is too lumpy, and is clogging the
needle.

So, any ideas how i can remove particulate matter from my DNA?

The DNA is originally from a Qiagen prep, and so far i've tried spinning
it down and putting it through a 0.22 micron cellulose acetate spin
filter, but neither of these solve the problem.

How about putting it through a silica column, such as a QIAquick? In
theory, the particles should either get stuck in the membrane or get
eluted in the washes, before the DNA. However, i am aware that silica
fines can leach from the membrane, which might also clog my tip.

Would CsCl purification work? Presumably, particulates won't band with the
DNA.

I could perhaps filter through an even small spin filter - since my
construct is ~3 kbp, it might well go through a 500 kDa filter. This could
be quite expensive, though.

Perhaps a fresh DNA prep is the way to go!

Also, does anyone know what the typical diameter of a microinjection
needle is? I know they vary according to how you prepare them, but i don't
even know what order of magnitude i'm working at. All the information i
can find pertains to the needles used for injecting oocytes, which seem to
be much larger (several microns across).

Many thanks,
tom

--
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) [Only registered users see links. ]

Reply With Quote
  #2  
Old 11-29-2006, 02:01 AM
Nick Theodorakis
Guest
 
Posts: n/a
Default Cleaning up DNA for microinjection


Tom Anderson wrote:
....

I haven't injected DNA, but I have injected antibodies into cells year
back. We used to give them a brief (about 20 min) spin at about 100,000
X g in a Beckman Airfuge (sort of a Barbie-sized ultracentrifuge driven
by compressed air).

Nick

--
Nick Theodorakis
[Only registered users see links. ]
contact form:
[Only registered users see links. ]

Reply With Quote
  #3  
Old 11-29-2006, 03:01 AM
DK
Guest
 
Posts: n/a
Default Cleaning up DNA for microinjection

In article <1164765694.610282.124080@80g2000cwy.googlegroups. com>, "Nick Theodorakis" <[Only registered users see links. ]> wrote:

Standard practice in many techniques. Spin down everything insoluble
and aggregated. Do they still make Airfuges? The greatest thing about
airfuge is that its rotor levitates. Great idea that works exceedingly well!

DK
Reply With Quote
  #4  
Old 11-29-2006, 03:10 AM
Nick Theodorakis
Guest
 
Posts: n/a
Default Cleaning up DNA for microinjection


DK wrote:
....

It appears so. Or at least, they still market them:

<http://www.beckmancoulter.com/products/instrument/clincentrifugation/airfuge.asp>

Nick


--
Nick Theodorakis
[Only registered users see links. ]
contact form:
[Only registered users see links. ]

Reply With Quote
Reply

Tags
cleaning , dna , microinjection


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
~ SERVICE APARTMENT ~ Abernathy Racanelli Botany Forum 0 07-16-2009 09:37 PM
tank cleaning (Padnos.Beth@epamail.epa.gov) (Jocelyn) Heather S McAllister (hmcallis@Princeton.EDU) Zebrafish Forum 0 09-04-2008 05:43 PM
tank cleaning (Padnos.Beth@epamail.epa.gov) Heather S McAllister (hmcallis@Princeton.EDU) Zebrafish Forum 1 09-03-2008 06:02 PM
"Help" ATR Ge crystal cleaning Gina Kandilioti Chemistry Forum 0 07-22-2003 07:08 AM
cleaning basin, bathtub and shower walls w/o using standard cleaning agents sold at the stores. amanda Chemistry Forum 4 07-19-2003 03:15 AM


All times are GMT. The time now is 03:15 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 0.13423 seconds with 16 queries