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| 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. |
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| There is no answer at this time. |
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| 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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