how to clean DNA10KB- Molecular Biology

I tried to clean a restriction product >10kb before ligation by a gel extraction method (Qiagen gel extraction kit). But the concentration was too low to do a ligation. I used to clean restriction products using Qiagen PCR purification kit when the DNA <10kb, the concentration is very good. But I cannot find a Qiagen PCR purification kit for large DNA.

anyone knows how to clean DNA>10kb very appreciate your answers.

waiting online!!!

-mmdhuilin-

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You can just purify your DNA by phenol extraction and ethanol precipitation. There should be no problem for ligation reaction.

-pcrman-

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when I use the QIAGEN gel extraction kit to recover the fragment more than 10 kb, I will use 2 columns. each column is eluted by 50 &mu;l TE, and mix them in one tube and precipitate by ethanol, then dissolve it into 10~20 &mu;l TE, and check 1 &mu;l with a gel. if you make a right molecular ratio between the insert and vector, it's no problem.

I use this method to ligate for two times, one time the fragment is more than 12 kb, the other time is more than 15 kb, all succeed.

-xysun-

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Hi,

I agree with pcrman, ethanol ppt is your best shot. Melt the gel slice in saturated LiCl solution by heating to 50-55'C and then follow the instruction in Maniatis.

Also, if you insist using QIAGEN's kit, make sure you allow the TE (or EB) to "sit" in the silica for a few minutes. Sometimes I even elute twice using half the final elution volume each time, allowing the buffer to sit in the silica for at least 3-5 mins each time.

-InvisibleSurfer-

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:)QIAGEN gel extraction kit only can recover the plasmid fragment from 70 bp to 10 kb. If the plasmid fragment is bigger than 10 kb, I found that the amount of the binding plasmid fragment on the column is limited and nearly the same everytime, no matter how much I loaded before.

So I use two columns everytime, and this can make me get enough plasmid fragment for ligation.

-xysun-

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I used QiaExII kit purified 19kb, with a satisfied yield.

-mich_ard-

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QUOTE(mich_ard @ Jun 17 2004, 10:30 AM)

I used QiaExII kit purified 19kb, with a satisfied yield.

Could you tell me your protocol much clearly, I have used this Kit to purify the DNA fragments of 9-23 kb, but the recovered DNA just sheared into small ones.

Any advise should appreciated!

Zhong-Min Dai

-zhongmindai-

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QUOTE(zhongmindai @ Jun 19 2004, 11:10 PM)

QUOTE(mich_ard @ Jun 17 2004, 10:30 AM)

I used QiaExII kit purified 19kb, with a satisfied yield.

Could you tell me your protocol much clearly, I have used this Kit to purify the DNA fragments of 9-23 kb, but the recovered DNA just sheared into small ones.

Any advise should appreciated!

Zhong-Min Dai

just follow the instruction, and handle gently, nothing special.

mike

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-mich_ard-

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I use the Promega Wizard Plus DNA purification kit (mini, midi, or maxi) and it can purify DNA up to 20kb in size. It is not as clean as Qiagen's kits, but it does the job of retrieving your DNA very well.

-Squawks-

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it is really simple!

1- Divide into two columns each instead in one

2- Pick up the flow-through elution, and do the elution again in the same column. This helps in more recovery of your DNA which failed to be eluted the first time!

I did that many times, and it works good for me?

Good luck!

-hepatite-

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Increasing recovery is also achieved by heating elution buffer (or water if you elute in water, but watch pH).

-vairus-