Hi,
I was wondering if anyone can help me with microparticle bombardment protocol. When doing my postdoc I used to obtain integrated lines by gold particle bombardment. However, now in my lab I tried many times without success. There are some differences comparing with my old lab:
- I used to use the hepta adapter, and now I have the single adapter
- In general I used plasmids, and now we’re trying to generate lines out of a fosmid we modified by recombineering. Based on sequencing and PCR, looks like the unc-119 is integrated.
- I’m using now the 0.6 micron gold particles, as opposed to 1 micron gold particles I used to use (I bought the equipment used and came with those gold particles).
I was wondering if anyone is doing bombardments the same way I’m doing it now and could give me either a protocol or some advice.
Last question. When I purify the fosmid in EPI300 + Ara I obtained like 500ug of DNA starting with 200ml LB. Is that normal? Isn’t it too much? Based on nanodrop. Anyone tried purifying without a column? Phenol chloroform?
Thanks in advance!
Dani
Hello Dani,
Couple of things you could try:
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I am not sure what is the efficiency of your rescued lines. You might need to adjust the pressure of Helium for the single Adapter, also you could try to adjust the position of bombarded plate (up or down) in the instrument chamber. Since it is single channel adapter, keeping plate too close will only bombard fewer worms due to lesser spread area of particles and if the plate is full of worm (90mm plate), most worms will not be bombarded as shot particles will only hit in the center and therefore it will reduce the numbers of transformants per bombardment. You could also try to bombard an empty plate to see the spread of particles and accordingly put worms only in that area.
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Fosmids vs plasmids may not have big differences in term of getting transformed lines as long as you have intact fosmid to begin with. I would suggest using a maxiprep kit(Qiagen) to get better quality fosmid preps. Ethanol precipitation of DNA has been reported to give better transformation efficiency at least for microinjection. If it is not too much to do, you could try that as well (http://wbg.wormbook.org/2011/12/14/omission-of-rnase-and-addition-of-ethanol-precipitation-improve-transgenesis-using-spin-column-purified-dna/).
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I don’t know if the size of particles can influence the number of transformants. It might affect in term of less surface area to hold the DNA. You can also try 1 micron tungsten particles, they are cheap and has worked in our hands.
You could try to estimate the correct quantity of DNA by digesting it with restriction enzymes (which cut 2-5 places) and running it on Agarose gel and estimating the DNA of this fosmid by comparing to the known quantity of DNA ladder. You need ~500ng/ul concentration of DNA to coat the gold particle. We have found that Nanodrop can sometime overestimate the quantity of DNA due to presence of RNA or free nucleotides.
Hope this will help.
Hi Dani,
We definitely notice a difference between fosmids and plasmids in our lab. We do 12 bombardments per week to get transgenic fosmid and plasmid lines in the Waterston lab. We use the hepta adapter. If you want, I can give some more tips and send you our protocol if you email me: adwarner (at) uw.edu
Thanks Mainpal, tons of useful information!
I was wondering if you can comment on # of worms. I used to grow a mixed population of DP38 or HT1593 in 6 egg plates and transfer them to a 90 mm dish for bombardment with the hepta adapter. Lately in the lab we’ve been growing worms in egg plates and transfer each of them to a 60 mm dish for bombardment. We accommodate worms in the center of the dish (around 20-30 mm diameter). Do you do something similar?
Thanks!
Dani