
PCR product purification
#16
Posted 09 September 2004 - 05:23 AM
I use Jetsorb from Genomed. It works every time with small and bigger fragments.
Good Luck
#17
Posted 10 September 2004 - 11:38 PM
#18
Posted 14 September 2004 - 01:40 AM
#19
Posted 14 September 2004 - 06:45 AM
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
#20
Posted 14 September 2004 - 12:55 PM
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

#21
Posted 15 September 2004 - 07:58 AM
/Nina
#22
Posted 22 September 2004 - 11:58 PM
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
#23
Posted 23 September 2004 - 12:24 AM
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.
#24
Posted 25 September 2004 - 08:18 AM
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!!

#25
Posted 06 October 2004 - 05:11 AM
Sometimes the the oldies are the best!
#26
Posted 18 April 2009 - 02:57 AM
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!
#27
Posted 06 August 2009 - 10:30 PM
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.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#28
Posted 10 August 2009 - 05:55 PM
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.
#29
Posted 07 November 2009 - 06:00 AM
#30
Posted 16 July 2011 - 07:17 PM