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Removing EDTA from DNA buffer

  • 17-02-2014 10:29am
    #1
    Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    I have been using a few different methods for DNA extraction for different downstream experiments. Recently I have been looking at the old fashioned "dirty" alkali lysis method so that the DNA ends up immediately in a buffer after the lysis protocol eg Tris-EDTA buffer pH8. I am just wondering though, the equipment I wish to use the samples on seem to be extremely sensitive to the presence of EDTA (ie nothing works if EDTA is present) and I was wondering are there any ways of removing EDTA from a buffer (with the sample in it). The only thing popping into my mind is to just throw in some MgCl2, not that it removes the EDTA but hopefully it will interfere with the EDTA enough that it causes no downstream issues (I presume the EDTA is interfering with the downstream PCR chemistry).


Comments

  • Registered Users Posts: 621 ✭✭✭ cgc5483


    CramCycle wrote: »
    I have been using a few different methods for DNA extraction for different downstream experiments. Recently I have been looking at the old fashioned "dirty" alkali lysis method so that the DNA ends up immediately in a buffer after the lysis protocol eg Tris-EDTA buffer pH8. I am just wondering though, the equipment I wish to use the samples on seem to be extremely sensitive to the presence of EDTA (ie nothing works if EDTA is present) and I was wondering are there any ways of removing EDTA from a buffer (with the sample in it). The only thing popping into my mind is to just throw in some MgCl2, not that it removes the EDTA but hopefully it will interfere with the EDTA enough that it causes no downstream issues (I presume the EDTA is interfering with the downstream PCR chemistry).

    Why not just resuspend the DNA in 10 mM Tris; pH8


  • Registered Users Posts: 6,794 ✭✭✭ cookie1977


    Qiagen kits are cheap enough and their columns will strip out the salts. Do you not want to use a kit?


  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    We have Qiagen kits here but they are not that cheap for the number of samples I will be using. I am using an alkali lysis method which I originally was putting EDTA into to chelate anything that would help DNAses do their job before the heat step. Then my supervisor has informed me that the chemistry we are using has had huge issues in the past with any EDTA present.

    The buffer is formed from the Lysis buffer and the neutralising buffer added afterwards combine to create a Tris-EDTA buffer. I could leave out the EDTA and try and get it onto the heat step ASAP and hopefully the DNAses actions will be minimalised.

    The only other option barring something like the Qiagen clean up (which would defeat the purpose of the dirty lysis as I could just do a PK lysis if I was going down that route). the dirty lysis is purely to get a high volume at low prices through.

    I might just run a few test plates this week, one with no EDTA, one with EDTA plus an addition of MgCl2 once it is buffered and one with just EDTA and see if my super was wrong when it is such a low concentration (0.2mM).


  • Closed Accounts Posts: 4,042 ✭✭✭ Milani Better Stretcher


    In theory the addition of the Mg should occupy the EDTA alright. Remember each EDTA molecule will bind two Mgs so in theory you need to have double the MgCl concentration. I'd probably lump in ten times as much or so just to be safe if the MgCl isn't going to effect anything else in the system. I have no idea what the stability constants are like for that type of reaction but I'd say its available somewhere. What type of chemistry are you attempting downstream just out of interest?


  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    What type of chemistry are you attempting downstream just out of interest?
    The chemistry is KASP but it is done externally by industry partners. The EDTA does not interfere internally with my larger scale test reactions but on their dried down plates with miniscule volumes apparently any presence at all seems to stop the PCR from working but that could be that the volumes they had tested were to concentrated so I may send them a test plate and not tell them it has EDTA just to see will it get through.


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  • Closed Accounts Posts: 4,042 ✭✭✭ Milani Better Stretcher


    Yeah you could. I'd say the EDTA is chelating the magnesium at the pcr step thus making the polymerase inactive.


  • Registered Users Posts: 621 ✭✭✭ cgc5483


    Too much magnesium can also interfere with your PCR reaction.

    I don't get where you say the buffer is formed from the lysis buffer and the neutralising buffer. In fact what you probably have is that you have Tris and EDTA in the resuspension buffer before you lyse with NaOH and then neutralise with potassium acetate.

    Do you do any isopropanol precipitation? If yes then I would suggest you precipitate with isopropanol, wash the pellet with 70 % EtOH and then simply resuspend the pellet in 10 mM Tris; pH 8.

    Not sure it's a great idea to not tell them about the EDTA. If a collaborator did that to me I wouldn't be too happy.


  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    cgc5483 wrote: »
    Too much magnesium can also interfere with your PCR reaction.
    True, I am going to try and just hit it on the head concentration wise, ie, just enough to neutralise it with little/no overage.
    I don't get where you say the buffer is formed from the lysis buffer and the neutralising buffer. In fact what you probably have is that you have Tris and EDTA in the resuspension buffer before you lyse with NaOH and then neutralise with potassium acetate.
    I am using the Alkali lysis from Truett in Biotechniques "Preparation of PCRQuality Mouse Genomic DNA with Hot Sodium Hydroxide and Tris (HotSHOT)"
    Do you do any isopropanol precipitation? If yes then I would suggest you precipitate with isopropanol, wash the pellet with 70 % EtOH and then simply resuspend the pellet in 10 mM Tris; pH 8.
    I do but I don't want to do it here, I want to follow the paper closely and go straight from the lysis to the PCR reaction. The combination of the alkali lysis reagent (NaOH and EDTA) followed by the neutralising reagent(Tris-HCl) should leave me with a DNA solution, the buffer being roughly pH8/8.1 Tris-EDTA. Admittedly I am also concerned about the formation of salts within the buffer and the possible interference this may cause but will test it first. If it shows no promise, I will back to the PK lysis with a Qiagen clean up.
    Not sure it's a great idea to not tell them about the EDTA. If a collaborator did that to me I wouldn't be too happy.
    I am sending them samples for testing, they have no idea what is in them anyway, I am just looking at different techniques and buffers. They have no idea what is in any of my solutions, I just wasn't telling them, in case they would treat it differently, I can see how my post would give a different impression.


  • Registered Users Posts: 6,794 ✭✭✭ cookie1977


    Is it plasmid DNA? Could you try to precitate out the DNA and resuspend.


  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    cookie1977 wrote: »
    Is it plasmid DNA? Could you try to precitate out the DNA and resuspend.
    I want to keep the entire protocol as cheap/cheerful as possible. Where possible I want to avoid manipulation of the DNA product, even if harmless. I was just hoping there was a simple step to remove EDTA or at least render it inert as far as the PCR process is/was concerned.

    I will see how the ideas above go and throw up results next week (ie sans EDTA and with MgCl2).


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  • Registered Users Posts: 284 ✭✭ josey_whale


    Questions...

    1. What type of cell are you using - mammalian? bacterial? yeast? you might not even need to use alkali lysis... you could get away with simply boiling your cells. DNAse's - I wouldn't worry about them. I have often done colony PCR (add a loop full of cells directly into the PCR) and never had any issue. The initial 95C of the PCR is sufficient to lyze the cells to get the reaction going.

    2. How much starting material are you using - low (<10^6) or higher than this?

    3. What is the concentration of EDTA, after the reaction is re-suspended on the plate? EDTA does inhibit PCR, but it only starts to get serious at concentrations >1.5mM, so you could dilute your DNA... ie, because of the sensitivity of PCR, all you need is a few molecules in the reaction to get a result.

    So, as I understand it, you lyse your cells, the neutralise the buffer, and dry it down into a plate? Why not lyse your cells, neutralise it, dilute 1:100 with H2O and then dry the same volume down into the plate - you have 100-fold less gDNA in there but also 100-fold less EDTA.

    4. Why do you need EDTA in there in the first place? lyse in NAOH, then neutralise, and then dry down?


  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    Questions...

    1. What type of cell are you using - mammalian
    Mammalian
    2. How much starting material are you using - low (<10^6) or higher than this?
    Varies sample to sample, they are not normalised and we use tissue so no cell count.
    3. What is the concentration of EDTA, after the reaction is re-suspended on the plate? EDTA does inhibit PCR, but it only starts to get serious at concentrations >1.5mM, so you could dilute your DNA... ie, because of the sensitivity of PCR, all you need is a few molecules in the reaction to get a result.
    0.1mM which is low but apparently any at all inhibits their system.
    So, as I understand it, you lyse your cells, the neutralise the buffer, and dry it down into a plate? Why not lyse your cells, neutralise it, dilute 1:100 with H2O and then dry the same volume down into the plate - you have 100-fold less gDNA in there but also 100-fold less EDTA.
    Was also planning this, gonna check the concentration on random samples and if high then dilute them down ( hopefully overcoming any salt fears as well).
    4. Why do you need EDTA in there in the first place? lyse in NAOH, then neutralise, and then dry down?
    For the DNAses but that's it.


  • Registered Users Posts: 284 ✭✭ josey_whale


    You could heat inactive the DNAse's in the sample 75C for 10mins should do it.

    So, I would take out the EDTA.... alkali lysis, neutralise with HCl, heat to 75 for 10mins.... dry down...


  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    You could heat inactive the DNAse's in the sample 75C for 10mins should do it.
    Can just leave out the EDTA altogether then, the Alkali lysis has a heat step for an hour, my concern was the DNAses would start acting straight away but if I placed the samples on while the Alkaline buffer was already on the heat, then that may just do the job for me.


  • Registered Users Posts: 284 ✭✭ josey_whale


    yeah, that would be my reference point.... build from the bottom.... I'd be interested to know how you get on.


  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    So I lysed two batches of samples (94 samples + 2NTCs each). The first I made a slip up with the neutralising buffer, not far off what it should have been and the second as per description before. No EDTA in either.

    The first run (based on Qubit dsDNA testing) contained between 0.05ng/ul and 14.5ng/ul of dsDNA based on a sub sample of 8 randomly selected samples.

    The second run came back better with concentrations between 2.97ng/ul and 26.8ng/ul of dsDNA based on a sub sample of 8 randomly selected samples.

    In both cases the low samples were ones that had been identified as fatty samples before the lysis.

    I should have the results for the PCR from these back by tomorrow or Friday to see whether they amplified correctly or not. Don't expect much back from the first batch but hopefully the second ones will be good.


  • Registered Users Posts: 284 ✭✭ josey_whale


    Good stuff. What volume is your final DNA in? You said it was tissue samples that you were using. Any idea of the weight of the sample you are extracting from?

    Provided, your DNA is quite clean, you should have loads of DNA there for PCR. Is it real-time PCR you are using?


  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    Good stuff. What volume is your final DNA in?
    keeping about 100ul in a PCR plate for storage reasons.
    You said it was tissue samples that you were using. Any idea of the weight of the sample you are extracting from?
    Varies sample to sample, I would hazard a guess that they are maximum 200ug but with many way below that. i have to lyse them straight off the collection device so I can only visually inspect them.
    Provided, your DNA is quite clean, you should have loads of DNA there for PCR. Is it real-time PCR you are using?
    Alas the first set of results came back and they are pretty awful. There is DNA there but it is obviously not good enough for the PCR they are doing. No amplification at all but I haven't went through all the results. Back to the drawing board.


  • Registered Users Posts: 884 ✭✭✭ Dingle_berry


    CramCycle wrote: »
    Alas the first set of results came back and they are pretty awful. There is DNA there but it is obviously not good enough for the PCR they are doing. No amplification at all but I haven't went through all the results. Back to the drawing board.

    Couldn't you dilute out the EDTA with a few washes?


  • Closed Accounts Posts: 4,042 ✭✭✭ Milani Better Stretcher


    If its real time PCR my heart goes out to you, such a **** of a technique to get right.


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  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    Couldn't you dilute out the EDTA with a few washes?
    I did want to avoid as many extra steps as possible as I wanted to keep the costs down and the through put as high as possible but I may not have a choice in the matter. A
    If its real time PCR my heart goes out to you, such a **** of a technique to get right.
    Ha, I know, done it for years, once you get it working right it is beautiful, its just getting it to that point. This isn't real time, but they are using flourophores/probes (SNP genotyping/KASP chemistry), so after x cycles they stop, read it, and then go for another 3 cycles, read it again and on and on and on. Then they just use whatever looks best.


  • Registered Users Posts: 32 Squeakf99


    CramCycle wrote: »
    I did want to avoid as many extra steps as possible as I wanted to keep the costs down and the through put as high as possible but I may not have a choice in the matter. A

    Ethanol precipitation is relatively quick and cheap but it is another unwanted step.

    1. Prepare 3M Sodium acetate (NaOAc) solution.
    2. Add 11µl NaOAc for every 100µl DNA.
    3. Add 2 X DNA volume of ice cold ethanol.
    4. Precipitate for at least 30 mins at -20°C.
    5. Centrifuge for 10 mins at 14,000g.
    6. Pour off the ethanol and wash the sides of the tubes with ice cold 70% ethanol.
    7. Centrifuge for 10 mins at 14,000g.
    8. Pour off the ethanol and allow to air dry until all the ethanol has evaporated.
    9. Resuspend

    Keeping the mixture on ice as well should keep the DNAses inactive at least partly


  • Moderators, Sports Moderators Posts: 22,722 Mod ✭✭✭✭ CramCycle


    Squeakf99 wrote: »
    Ethanol precipitation is relatively quick and cheap but it is another unwanted step.

    Do it all the time, still one of the best methods IMO if done with care. I have sent of some more samples for them to test. Using the same protocol as before but using additives as well that will hopefully both aid lysis/cell disruption as well as work as a PCR additive/booster. This time I have tried Tween 20.


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