49 replies to this topic

[haui]

Hi Guys,

I use Jetsorb from Genomed. It works every time with small and bigger fragments.

Good Luck

[iamfat]

for PCR product purification used for cloning, phenol/chloroform's classical method is ok. I think kits are unnecessary.

[Sphingoman]

I use the Qiaquick PCR purification kit all the time without having any troubles. Maybe your PCR did not work. Otherwise you can use a PCR puirification kit from Roche.

[Nina]

First look on agarose gel (2%) that you have PCR product of interest.

If you have low concentration you can pool some samples together (same kind of PCR product of course )

I normally use the PCR purification kit (Jet quick) from genomed Incorporation. I thinkl that one is working just fine.

I have purified PCR product that is 150bp.

I hope that you soon will purify youre sample succesfully.

/Nina

[turtle]

Don't forget that many of the kits elute the purified PCR product in water or buffer. This generally dilutes your product, especially for gel bands! 100 ng of DNA will give a nice, bright band if the initial well in the gel wasn't very wide. Bind that 100ng to a membrane and elute into 50 ul of buffer, and you've got a concentration of only 2 ng/ul! Load 5 ul on a gel to check for product/quantify, and you probably won't see a thing. Eluting in less than 50 ul increasaes your concentration, but you lose some product in the process.

So, if you don't see product after cleaning, and think you've lost your product, my suggestion would be to ethanol precipitate, preferably with a co-precipitant such as PelletPaint (Novagen). Then you can see if you have a product, cause if you don't you also won't have a pellet, and you can get your concentration back up to where you can see it on a gel or quantify it by spec.

Just my 2-cents worth.
-Turtle

[Nina]

I ususally purify three band, from the agarose gel containing the same PCR product, together to minimize the risk for diluting the sample.

/Nina

[julianne]

seeing that your clock was ticking since april, not sure how much help my reply will be..

anywayz,

been using sod acetate- isoprop precipiation with no problems all the while.

good enough for cloning and gives good seq result when used as template..

good luck!

let me know if u need the procedure

[postdoc]

for PCR product purification used for cloning, phenol/chloroform's classical method is ok. I think kits are unnecessary.

For cloning of PCR products, does phenol/chloroform extraction necessary? or can I just do a precipitation?

My two cents: With using a kit, to increase concentration while get good recovery, I usually use 25-30 ul buffer to elute DNA, after elution, use the same elutant to elute the same column once more.

[thegradstudent]

Thanks all you guys for your numerous replies!!!
I have just repeated the PCR and obtained a better band... Next time I have a pool of choices here!!! But I am absolutely sure that this helped a lot of people. That makes this site great!!

[tuckern]

Sounds like good old fashioned ethanol precipitation would do the trick.
Sometimes the the oldies are the best!

[OA17]

Hi!
I have also used QiaQuick PCR purification kit, and I didn't have problems. While using this protocol (as well as MinElute protocol, which actually works as well), I always centrifuge 2 minutes, instead of 1 minute, and when I add the water to elute DNA from the column I leave the column stand for 5 minutes (instead of 1 min), and that improves the final yield (at least for me).

For gel extraction y usually use the Freeze and Squeeze protocol, if the final goal is to clone the band. For remaplification, I would try not to go through gel (I have been having problems with this, but I guess I should ask people at the forum).

¡Saludos!

[Adrian K]

Is the Wizard DNA clean-up kit well?
I'm trying!

I had been using Promega Wizard PCR clean up kit. It works well for me. But for the elution, I usually elute ~65% volume from my initial volume which I had loaded into the gel.

[jiajia1987]

Hi!
I have also used QiaQuick PCR purification kit, and I didn't have problems. While using this protocol (as well as MinElute protocol, which actually works as well), I always centrifuge 2 minutes, instead of 1 minute, and when I add the water to elute DNA from the column I leave the column stand for 5 minutes (instead of 1 min), and that improves the final yield (at least for me).

For gel extraction y usually use the Freeze and Squeeze protocol, if the final goal is to clone the band. For remaplification, I would try not to go through gel (I have been having problems with this, but I guess I should ask people at the forum).

¡Saludos!

For my case when it comes to QIAqiuck PCR purification kit, I always centrifuge for 1 min and I always let the column stand for a longer period of time (just like you). In addition, sometimes I will heat the water for elution up to 50degcel before I use it for elution. Improves the yield too. Increasing the binding buffer also helps.

[pop09]

I use shrimp alkaline phosphatase and Exo I. I have a mix that I put in my PCR tube, and do the reaction in a the PCR machine. I don't reduce my product and it works very well for sequencing (I haven't tried other downstream applications with it).

[sciencelover]

I recommend using the [url="[url]http://www.alkaliscientific.com/en/gel-dna-recovery-kits/697-zymoclean-gel-dna-recovery-kit.html"]Zymoclean[/url] gel recovery kit[/url] for the gel extraction and one of the [url="[url]http://www.alkaliscientific.com/en/pcr-dna-clean-up-concentration-kits/689-dna-clean-concentrator-5.html"]DNA[/url] Clean-up and concentration kits[/url]. I used to use Qiagen and I switched to these because they have much better yields.