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digestion & purification problem


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#1 netnus

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Posted 01 October 2004 - 09:52 AM

I am trying to ligate a 500bp insert to the 4K pET vector with restriction sites BamH and Nde1. To increase the cleavege efficency I have to sequentially digest the insert and vectro which means I ought to change the buffer before I do the second digestion.
My colleagues sugguest me to gel-purify it. I am really uncomfortable to gel-purify DNA using all kinds of gel purification kits. They says the recovery efficiency will be about 60%. but In fact it is far less than 60%.
Any suggestion will be greatly appreciated!


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#2 mario2004

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Posted 01 October 2004 - 04:07 PM

You don't need to change buffer. You can start with the enzyme which requires low salt buffer, after the first disgestion, then adjust the salt concentration of the reaction (using a small volume of a concentrated salt solution) to approximate the reaction conditions of the second restriction endonuclease. If low recovery is a concern, just do a phenol extraction and ethanol precipitation.

#3 BryanW

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Posted 02 October 2004 - 09:37 PM

If the commercial gel extraction kits only give you less than 60% recovery, for your purpose it still enough for ligation. Gel purification kits did do a great job to remove unnecessary stuff which will greatly enhance ligation/transformation efficiency. And it will save you time....
Our lab use Epoch Biolabs' gel extraction kit (epochbiolabs.com) with ~80% recovery and the purified fragments are excellent for downstream ligation recation.

#4 kant0008

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Posted 04 October 2004 - 10:55 PM

Hi,
yeah, i agree with mario, you can ethanol precipitate, but I find that you tend to lose more DNA than with gel kits. Also, less than 60% recovery is not too flash for a kit, which one have you used? I use Qiagen gel kit, and it's ok, although I must say that the smaller fragments (abut 100bp) are lost at a much higher rate- but for you that should be ok. Also sometimes you need to optimise the protocol of the kits just a little bit, eg incubate with elution buffer for longer.
Good luck

#5 netnus

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Posted 05 October 2004 - 04:08 PM

Hi,
yeah, i agree with mario, you can ethanol precipitate, but I find that you tend to lose more DNA than with gel kits. Also, less than 60% recovery is not too flash for a kit, which one have you used? I use Qiagen gel kit, and it's ok, although I must say that the smaller fragments (abut 100bp) are lost at a much higher rate- but for you that should be ok. Also sometimes you need to optimise the protocol of the kits just a little bit, eg incubate with elution buffer for longer.
Good luck

I use bio-rad Quantum Prep Freeze N Squeeze DNA Gel Extraction Spin Column. This spin column requires nothing. Only thing you need to do is to cut out the bands from the gel and chop the gel slices and place them into the columns and spin it down. It is very quick but I don't know it is efficient. Here I would give you an example:

Original DNA con: 31ng/ul 40ul
Con after extraction: 3ng/ul. 60ul

which means the efficency=3*60/(31*40)=15%

Anyone else uses this column to recovery the DNA? If so, how efficient the column is? Is there any trick while you recover the DNA?

#6 monolithis

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Posted 23 March 2005 - 09:28 PM

Hi,
I have used Biorad's freeze n squeeze often and it has given me excellent yields. In fact, it is one of my lab's favorite systems/kits.

A good idea is to chop your gel into smaller pieces before you freeze them, and then, spin it down for 4-5 minutes.

You might want to concentrate your DNA (Microcon columns) since it gets eluted in about 100-150uL liquid depending on the size of your gel piece/s. Concentrating my DNA has helped me for my ligations and quikchange mutagenesis.

A nice advantage in this method is that it avoids the use of any chemicals like QG buffer (which has Iodine), propanol, etc. that the Qiagen kit employs. Freeze n' squeeze is a physical process that sucks your DNA down through the column along with the liquid from the gel.

Good luck!

#7 Khazaey

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Posted 24 March 2005 - 05:23 AM

Hi
I think you don'n need to change the buffer, I used these two enzyme before together, Which buffer do use? I suggest to use Tango buffer ( 2x yello buffer of fermentase company)




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