11 replies to this topic

[NiceButDim]

Hey guys, just a very quick one if I may

I'm double digestion my vector (working well)

Purifying it on a gel

Excising the appropriate band (get lovely bands)

Normally the next step would to be do a gel extraction but for some reason the kits are giving me very very low yields (almost nothing).  Have decided to maybe go back to basics and try a phenol-chloroform extraction and see what the problem is.

My question to you is, since my sample is purely DNA, can I skip the phenol-chloroform part and simply jump straight to the ethanol precipitation?  Do I just dissolve my excised band in water and proceed with the precipitation or do I need to dissolve my sample in a particular buffer to avoid interference from the agarose?

Many many thanks for anyone able to help, really very much appreciate it guys.

Edited by NiceButDim, 10 February 2009 - 12:59 PM.

[pcrman]

First, what kit did you use for gel purification? You can use Zymo gel purification kit which gives you very concentrated DNA.

If you just want to concentrate your DNA from your gel purification, you can go ahead doing ethanol precipitation.

If your DNA is still in the gel, you need first to get the DNA out of it by spinning (puncture a hole in the bottom of a 0.5 ml tube using a needle) or some other ways and then precipitate your DNA.

[NiceButDim]

pcrman, on Feb 10 2009, 11:30 AM, said:

First, what kit did you use for gel purification? You can use Zymo gel purification kit which gives you very concentrated DNA.

If you just want to concentrate your DNA from your gel purification, you can go ahead doing ethanol precipitation.

If your DNA is still in the gel, you need first to get the DNA out of it by spinning (puncture a hole in the bottom of a 0.5 ml tube using a needle) or some other ways and then precipitate your DNA.

I've used a QIAquick gel extraction kit (50) and QiaQuick spin columns (50).  I'm trying to elute a sample around 9 kb in size and think this kit works up to 10 kb if I remember.  Have also used a similar kit for larger sample sizes as initially I thought that might be the problem but no joy there.

I might try the ethanol precip. first as we have the reagents here to do it but will keep the Zymo in mind thank you.

Regarding the last part of you comment.  My DNA sample will indeed still be inside the gel but I'm not entirely sure I follow you regarding the punching the hole thing.  I thought I'd be able to just dissolve the agarose gel containing my DNA in a buffer (50 oC) and then proceed to precipitate as per normal?  Thats not the case no?


(edit: just noted that in my originally post I mentioned using a "miniprep" kit, that was just a typo apologies.  Edit out now)

Edited by NiceButDim, 10 February 2009 - 01:02 PM.

[bob1]

As agarose is a sugar based compound, just dissolving the agarose is generally not enough, it will precipitate in the extraction proceedure along with the DNA.  Ideally you will remove all or most of the agarose from the extraction before precipitation.

PCRman was alluding to this in his post - put as small hole in the bottom of a 0.5 ml tube and place your gel slice in it, then put the 0.5 ml tube into a normal eppendorf and centrifuge out the liquid from the gel.  As DNA doesn't bind to the agarose, it is still in solution and should come out of the gel with the electrophoresis buffer.

Having said all of this - I have found that kits work far better than all of the other proceedures I have tried, so I would persevere with the kits.

[swanny]

We found lots of troubles with the qiagen kit unless we were using absolutely top-grade agarose. In the end we started using the Promega kit for PCR / gel cleanup and yields jumped through the roof.

You could use low melt agarose, then extract by heating the band until the gel melts, then do the EtOH precip. Na acetate has worked well for me with this.

[scolix]

Try Invitrogen kit, they seem to be giving me good yields

[NiceButDim]

Thanks for all the feedback here guys, very helpful.
I went ahead with the ethanol precip. by simply dissolving the agarose and came a cropper with the agarose sugar later on in the procedure.  Punching a hole in the bottom and filtering off the agarose that way makes a lot more sense and something I will try next time.

The thing with the kits is, I don't think it's a problem with the kit rather then something I'm doing wrong with it.  Someone else in the lab used it for the exact same experiment (same vector, same digestion, same gel extraction) and had no problems.  I'm trying to pinpoint with them what I'm doing wrong but in the mean time I'd like to try and have a back-up working just in case.    

Again many thanks guys.

[swanny]

NiceButDim, on Feb 13 2009, 03:14 AM, said:

Thanks for all the feedback here guys, very helpful.
I went ahead with the ethanol precip. by simply dissolving the agarose and came a cropper with the agarose sugar later on in the procedure.  Punching a hole in the bottom and filtering off the agarose that way makes a lot more sense and something I will try next time.

The thing with the kits is, I don't think it's a problem with the kit rather then something I'm doing wrong with it.  Someone else in the lab used it for the exact same experiment (same vector, same digestion, same gel extraction) and had no problems.  I'm trying to pinpoint with them what I'm doing wrong but in the mean time I'd like to try and have a back-up working just in case.    

Again many thanks guys.

The local qiagen rep suggested I heat the elution buffer to 65C before the final step. This did help a bit to increase yields.

[microgirl]

I get horrible results with the Qiagen kit also, but . . . it depends on what you want to do with your excised band. If you're trying to ligate it into something else, I've always been able to get plenty of colonies ligating negative quantifications of DNA from the Qiagen kit into a new vector! Good luck.

[hanming86]

What did you use to quantify the DNA after gel extraction. I notice that the reading by nanodrop is "masked" after gel extraction.

Another thing is that how did you do the gel extraction . For improved yield i typically modify the comb and make the well 2-5x bigger so eventually i will get to fill essentially 90% my digestion product in the well ( remember to start with high amount of DNA in the digestion part ) .10 % i used for normal well , just wanna take a nice picture  

[tyrael]

pcrman, on Feb 10 2009, 10:30 AM, said:

First, what kit did you use for gel purification? You can use Zymo gel purification kit which gives you very concentrated DNA.

If you just want to concentrate your DNA from your gel purification, you can go ahead doing ethanol precipitation.

If your DNA is still in the gel, you need first to get the DNA out of it by spinning (puncture a hole in the bottom of a 0.5 ml tube using a needle) or some other ways and then precipitate your DNA.

hi there. about the ethanol precipitation part,
is it just wash the DNA in ethanol for few times ? or is there any particular standard protocol for that ?

thanks

[swanny]

NiceButDim, on Feb 11 2009, 04:07 AM, said:

Hey guys, just a very quick one if I may

I'm double digestion my vector (working well)

Purifying it on a gel

Excising the appropriate band (get lovely bands)

Normally the next step would to be do a gel extraction but for some reason the kits are giving me very very low yields (almost nothing).  Have decided to maybe go back to basics and try a phenol-chloroform extraction and see what the problem is.

My question to you is, since my sample is purely DNA, can I skip the phenol-chloroform part and simply jump straight to the ethanol precipitation?  Do I just dissolve my excised band in water and proceed with the precipitation or do I need to dissolve my sample in a particular buffer to avoid interference from the agarose?

Many many thanks for anyone able to help, really very much appreciate it guys.

Something else to think about:
How large is the fragment you remove by digestion? If it's under ~100 bp, you can EtOH precipitate using ammonium acetate instead of sodium acetate (you also have to change the volumes slightly, use 1/2 volume 7.5 M NH4. acetate and 2 vol EtOH).
If you're going on to clone something into the vector, treat the precipitated vector with phosphatase before cloning; this will make sure that any pieces of the digested DNA that are hanging around cannot clone.

Wear your lucky socks, jocks, hair band or whatever works for you!