Posted 11 July 2012 - 09:42 AM
I'm trying to extract genomic DNA from Ganoderma boninense of 5 different strains, so as to use it for PCR with redundant primers to amplify micro-satellites, for purposes of observing diversity. Once optimised this will be used in the field. What I'm doing is more of an optimization.
Unfortunately this is my first independent stab at molecular techniques - my first taught or guided stab being in a module where we were supposed to do a RT-PCR, it failed from the offset ad the entire class spent the rest of the time filling out the coursework book "as if" it had worked - so I'm not 100% happy with it all.
I've done a CTAB as follows:
1). 100mg of mycelia added to 1.5ml tube (prepared by liquid nitrogen freezing and grinding in pre cooled mortar and pestle & stored at -80 degrees C).
2). 500 micro-litres of CTAB buffer added, incubated at 65 degrees C for 30 minutes.
3). 500 micro-litres of Chloroform :Isoamyl alcohol added. Mixed until homogeneous and then centrifuged in a micro-centrifuge (13,000 rpm) for 5 minutes.
4). Top layer extracted to a new tube - avoiding the lower layers. Carried out the Chloroform step again.
5). Transferred top layer again, added 100 micro-litres/ml of RNase A, incubated at 37 degrees C for 30 minutes.
6). Added 0.54 volumes of chilled Isopropanol, left at -20 degrees C overnight to precipitate.
7). Centrifuged at 13,000 rpm for 40 minutes afterward I could see, some smear on the bottom I took to be the DNA.
8). Removed supernatant, avoiding disturbing the bottom pellets/smears.
8). Added 100 micro-litres of chilled 70% ethanol, flicked & inverted to mix, spun again at 13,000 rpm for 5 minutes.
9). Removed ethanol avoiding pellet, added 100 micro-litres of TE buffer, flicked and inverted to mix.
I'm sure I saw precipitate as smears/pellets at the bottom of the tubes, but when I ran 5 micro-litres of the stuff with 2 micro-litres loading dye on a 1% agarose gel at 50volts I didn't get any visible bands, the ladder I loaded did work but wasn't all that vibrant either.
Does anyone have any ideas or advice? am I doing something wrong? I'm using autoclaved pipettes and doing most of this in a fume hood because of the chloroform:isoamyl alcohol. I'm going to try again with fresher samples and a new CTAB buffer, but other than that I can't imagine why it's not working, I know some people heat the CTAB but many other papers out there don't bother, or at least don't state it. I'm wondering if centrifuging is too long at it's meaning the stuff at the bottom isn't re-suspending.
Sorry for the long post - I'm working to optimize this RAMS protocol from a past student's thesis, and they left some key details out. In fact in the study that was published from the part of the thesis I'm working from it says a commercial kit was used. In the thesis it states some of it was kit, some was CTAB.
Posted 11 July 2012 - 03:27 PM
Isopropanol pellets are usually clear or white (some protein carry over usually) and gel-like, and should be attached to the side of the tube near the bottom, instead of being directly in the bottom. If you place all the tubes in the microfuge with the hinge side to the top, the will always know where the pellet is/should be - directly down from the hinge, near the bottom of the tube.
- Axolotl9250 likes this
Posted 12 July 2012 - 12:16 AM
Other ideas for trouble shooting:
first of all are you using a fungal culture (i.e. mycelium from petri dishes) or fruiting bodies? There often is an enourmous difference in the outcome of DNA extraction depending on the material used.
second can you tell us what is in your CTAB Buffer - there are a lot of slightly different recepies around - which might influence your results
I would think about including an Acetat (NH4 works best for me, but also Na, or K Ac do the job) step at some point - to remove Polysaccharides etc which often are a problem when extracting DNA from fungi (also aids the DNA preciptiation later on)
You are using a considerable amount of fungal material - so you will have quite some protein carry over - as already pointed out by Bob1, but you should get a clearly visible band on an agarose gel
- best probably is to start again and monitor every step critically, because when you are new in the lab sometimes things go wrong without you noticing it because there are too many things to care about at once - and when you do it a second time suddenly everything works fine
keep us informed about your progress
Posted 12 July 2012 - 01:54 AM
20g of CTAB powder
280ml of 5M NaCl.
40ml of 0.5M EDTA pH8.
100ml of 1M Tris-HCL pH8.
Made up to 1 Litre with ddH20 (autoclaved). All mixed in a sterilized bottle and flea.
Then when I want to use it I transfer about 50ml to a plastic, pre-sterilized tube and add 1% 2-mercaptoethanol.
I'm using a mycelium that was growing in Potato Dextrose Broth for 2 weeks, since Ganoderma is a slow grower, then using sterile filter paper and funnels to remove the broth, then washing with autoclaved distilled water. Then I take the mycelium and grind in liquid nitrogen in a pre-cooled mortar & pestle. I made sure the material didn't thaw.
One method I've seen did use 1:5 v/v ammonium acetate (I think of 7.5M, but it wasn't specified) added to the aqueous supernatant after a second chloroform:isoamyl alcohol step, during precipitation with 2:1 v/v absolute ethanol and then 500 micro-litres of 0.2M ammonium acetate with a second 2:1 v/v absolute ethanol precipitation. I left this out though as the method I emulated this time did not feature it.
Do you have any recommendations as to the amount of material I should be using? Most methods I'm reading just say "material", I'm wondering if there's some kind of rule of thumb - so much material for such and such a working volume or something like that.
I'm currently harvesting fresh material now as the stuff I've been using is a little older than 2 weeks now. My lab partner found a protocol he thinks worked quite well and is going to give me the details shortly when he gets a break. I'll update when I get it.
Edited by Axolotl9250, 12 July 2012 - 01:57 AM.
Posted 12 July 2012 - 06:46 AM
How much fungal material are you able to harvest from each flask? Because if the 0.1g is all the material you harvest after two weeks, I would think about either using agar plates or using a longer incubation time to get more starting material - the wet weight of your culture is much higher than the amount of fungus.....
The buffer sounds alright, I usually omitt the ßMercaptoethanol - because it had adverse effects on the DNA extraction efficiacy when I used it with fungal cultures, especially basidiomycetes.
Posted 12 July 2012 - 07:34 AM
Posted 13 July 2012 - 03:24 PM
Posted 15 July 2012 - 03:52 AM
Posted 15 July 2012 - 11:51 PM
Have you tried to run a gel to see how your DNA looks like?
Have you tried a reference PCR with your samples (i.e. ITS primers) - and if did this control reaction work? Such a PCR tells you if your DNA is ok or if you have a probem with your DNA extract.
Too much DNA is often a problem esp when using microsatellites (it was in my hands) - I would start my troubleshooting by trying to dilute the samples.
- Adrian K likes this
Posted 16 July 2012 - 07:18 AM
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
Posted 16 July 2012 - 07:28 AM
Posted 19 July 2012 - 01:56 AM
Posted 20 August 2012 - 04:06 AM
1). Add 100mg of flash-frozen ground mycelium to 500 micro-litres of CTAB buffer, add 10-20 acid-washed glass beads & vortex for 45 seconds.
2). Incubate in a water bath at 65 degrees for 40 minutes, invert several times during.
3). Let the tubes cool to room temp before adding 2 micro-litres of RNaseA and incubating at 37 degrees for thirty minutes.
4). Add 500 micro-litres of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) and vortex until the phases are not distinct.
5). Centrifuge for 10 minutes at 4000rpm.
6). Carefully remove the aqueous supernatant to a fresh tube. Add 700 micro-litres of isopropanol. Invert once to mix.
7). Put tubes at -20 degrees for 2 hours.
8). Centrifuge at 13,000rpm for 20-30 minutes.
9). Remove supernatant & wash with 70% ethanol twice.
10). Make sure all ethanol is removed and re-suspend pellet in 50 micro-litres of TE buffer.
I have reasonable success with this and the yield is fairly good, however the isopropanol precipitation was at -20 degrees overnight because I was caught up in another part of my work. I'm concerned a few less desirable purity values may be caused by the fact that isopropanol at -20 that long will cause other things to fall out of the solution. If I wanted to solve this I see two possibilities, first keep the isopropanol precipitation at room temp for two hours instead, eliminating the need for -20 degrees. Second I can try ethanol precipitation although I need to know whether 2 volumes of 70% ethanol is adequate. I've seen protocols that use abs. ethanol and some also add a certain amount of ammonium acetate e.g. 50ul in one protocol or 1:5v/v of 7.5M to give a 1.5M conc. in the tubes in another. I'd also like to know how people manage a pellet of DNA which is being stubborn and not dissolving into TE at the end? I did my steps on friday and quantified DNA then with nano-drop but I noted my tips would sometimes get a bit clogged because of the pellet. It may have dissolved more over the weekend. I've seen a suggestion to put tubes at 60 degrees and it speeds things up, but they didn't say how long for or if it was possible to do it too long.
Posted 20 August 2012 - 04:30 AM
Posted 21 August 2012 - 08:24 AM
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