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Q: troubleshooting coating Gold particles with DNA ( No Answer,   4 Comments )
Question  
Subject: troubleshooting coating Gold particles with DNA
Category: Miscellaneous
Asked by: vishvaskulkarni-ga
List Price: $2.00
Posted: 15 Apr 2005 13:35 PDT
Expires: 15 May 2005 13:35 PDT
Question ID: 509798
I have been working for biolistic transformation of plant tissues with
DNA coated gold particles. Unfortunately, always have a doubt about
quality of Gold particle preparation.

Does there exist any simple test to check whether the Gold coating
with DNA was proper (before bombarding)?

I am also looking for reviews or detailed write-ups focused on the theme -
"troubleshooting / factors controlling" the "efficient coating of Gold
particles with DNA" for biolistic transformation of plant tissues.
Answer  
There is no answer at this time.

Comments  
Subject: Re: troubleshooting coating Gold particles with DNA
From: politicalguru-ga on 17 Apr 2005 10:05 PDT
 
Thank you for your question.  
 
However, I believe that to answer it well, your question will require
more time and effort than the average amount of time and effort
associated with this price. Here is a link to guidelines about pricing
your question, in the pricing guide:
https://answers.google.com/answers/pricing.html
Subject: Re: troubleshooting coating Gold particles with DNA
From: quantumdot-ga on 19 Apr 2005 13:15 PDT
 
What I can gather is you are 

1) Making gold nanos
2) functionalizing them with thiolated DNA
3) shooting them into the cells for some reason

and you want to know if 1 and 2 are working

EASY!

If you've made it right, you wont see any aggregation as you increase
the saline concentration of the solution. IE, your soln will stay
pink. If thats not enough, incubate with the complement to the ssDNA
you functionalized the nanos. The solution should turn purple as the
DNA hybridizes, and the localized surface plasmonns of the nanos
couple, and shifts to longer wavelengths. This is reversible
aggregation, and can be de coupled by heating the solution above Tm,
and then centrifuging the particles, or running them on a gel.

On a side note, I'd like to know more about the project. EG, im not
really clear on what biolistic transformation of plant tissues means.
However, if Im not on the right tack, set me straight, and Im sure I
can answer the question.
Subject: Re: troubleshooting coating Gold particles with DNA
From: vishvaskulkarni-ga on 29 Apr 2005 12:45 PDT
 
No. I am not making Gold nanos.

It is simply the coating of readymade Gold particles (o.6 micron, Bio
Rad make) and coating these with plasmid DNA carrying a gene to be
expressed in plant cells.

The procedure uses Gold particles, DNA of interest, Calcium Chloride
and Spermidine. The mixture is suspended in Ethyl Alcohol and spread
on a membrane which is biolistically shot into plant cells.

There is no Thiolation.

The problem is to assure that everything (Gold coating with DNA, and
plating on membrane etc.) was appropriate before shooting it.
Subject: Re: troubleshooting coating Gold particles with DNA
From: quantumdot-ga on 04 May 2005 11:44 PDT
 
This is for a Helios gene gun? I belive biorad has some protocols on
the site, but of what utility they may be, I dont know. What maybe
more usefull is to google (or scifinder) biolistic transfection, track
down an author from a paper in a reputable journal, and talk to the
head grad student or post doc.

I tried looking up info on the mfgr. of the particles (600 nm in HUGE
compared to what I'm used to seeing!) and couldn't find anything, but
I'm going to assume that they are using electrostatics to stick the
oligo (in your case, full plasmid) to the gold.  That should keep it
on there fairly tight. You could probably centrifuge or run a gel to
see if the plasmid is on the particles. Once its on the membrane, I
suppose it *could* come off, but its not likely to (not in ethanol
anyway). The only good test after that would be to actually do the
shoot and try a PCR, which defeats the purpose of the question.

I'll snoop around and see if I can come up with anything better.
QD

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