3 replies to this topic

[albatross]

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!

Edited by albatross, 01 September 2009 - 07:43 AM.

[almost a doctor]

albatross, on Sep 1 2009, 04:41 PM, said:

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!

How are you purifying your products?  It'll be easier to suggest options if we know what  you are doing.

[albatross]

almost a doctor, on Sep 1 2009, 07:53 AM, said:

albatross, on Sep 1 2009, 04:41 PM, said:

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!

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.

Edited by albatross, 03 September 2009 - 07:14 AM.

[gebirgsziege]

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!!!)