PCR product purification - (Sep/01/2009 )
I often encounter a problem during PCR product purifications:
The desired fragment is always contaminated with small amount of unexpected fragment, particularly smaller ones.
For example, when I PCR amplified tDimer 2 gene which is about 1.4 kb, the purification pruduct is contaminated with a 0.7 kb fragment. This may because of the tandem repeat of tDimer 2 gene. However,
I found this problem happen a bit frequently when purify other PCR products.
Any consideration?
Thank you!
albatross on Sep 1 2009, 04:41 PM said:
The desired fragment is always contaminated with small amount of unexpected fragment, particularly smaller ones.
For example, when I PCR amplified tDimer 2 gene which is about 1.4 kb, the purification pruduct is contaminated with a 0.7 kb fragment. This may because of the tandem repeat of tDimer 2 gene. However,
I found this problem happen a bit frequently when purify other PCR products.
Any consideration?
Thank you!
How are you purifying your products? It'll be easier to suggest options if we know what you are doing.
almost a doctor on Sep 1 2009, 07:53 AM said:
albatross on Sep 1 2009, 04:41 PM said:
The desired fragment is always contaminated with small amount of unexpected fragment, particularly smaller ones.
For example, when I PCR amplified tDimer 2 gene which is about 1.4 kb, the purification pruduct is contaminated with a 0.7 kb fragment. This may because of the tandem repeat of tDimer 2 gene. However,
I found this problem happen a bit frequently when purify other PCR products.
Any consideration?
Thank you!
How are you purifying your products? It'll be easier to suggest options if we know what you are doing.
I used the Qiagen purification kit.
Another problem is that i can not remove the primer dimer completely by gel purification even though the fragment is very large, 2.5 kb for example.
This used to be on protocol online.....
PEG purification
I only use this protocol, its easy robust, cheap and better than all kits I tried. Primer dimers are completely removed (but all fragments smaller than 200 bp as well!!!)