[Q] How to purify small PCR frg. ?

QIAGEN QIAGEN at kaiwan.com
Wed Jul 19 16:33:08 EST 1995


In article <3uidfs$4cs at mserv1.dl.ac.uk>, p_rudolph at MORPH.SPACENET.DE (Peter
Rudolph) wrote:

> recently a coworker of mine ran into a problem:
> She created a library with PCR using randomized primers,
> then she digested the fragment and want to ligate it into a vector.
> The complete PCR fragment is 96bp, after digestion she gets
> two fragments: 29 and 67bp. She separated them by gel
> electrophoresis on a 2.5% AG.
> The problem is now: How to purify the bigger fragment from the
> gel slice with a really *high* yield?
> She tried freeze-and-squeeze and Qiagen-gel-ex but got
> only very low yields.
> Any suggestions?
> Peter.

Hi Peter and  Co-worker:

We  assume you have used QIAGEN's QIAEX Gel Extraction Kit. You've got low 
yield from using QIAEX because of the following possible causes:

-  Binding conditions: When binding the DNA to the QIAEX suspension, the pH

of the mixture of QX1 binding buffer + your sample + QIAEX suspension
should 
be less than 7.5. To be safe we even prefer it to be below 7.0. Re-using 
electrophoreses buffer can cause the pH to be too high--beyond the
buffering 
capacity of QX1. You can simply checkthe mixture  with a pH strip. If the
pH 
is too high, add 10 ul of 3M NaAcetate pH 5.0 to the mixture.

- For agarose gel >2%, such as in your case, make sure you double the
amount 
of QX1 binding buffer.

- Overdrying of QIAEX suspension during evaporation of ethanol containing
QX3 
Buffer. Overdrying causes the DNA to bind too strongly to QIAEX particle.  
Dry the pellet until you see a white rim around the edge of the pellet.
This 
step is one of the reasons why  we upgraded the QIAEX to QIAEX II, which
also 
provide higher yield.

- Elution conditions: contrary to binding conditions, pH of elution buffer 
should be greater than 7.5. We recommend 10mM Tris-HCl pH 8.5. If youelute 
with water, be cautious because water pHsometime is  acidic, although 
theoretically it should be neutral.

QIAEX was designed to sufficiently recover fragments from 40bp to 
50kb. Nevertherless, less than 50% of fragments smaller than 40 bp will 
be recovered. 

If you have any questions, please contact our friendly molecular biology 
specialists in Technical Service dept. at 800-DNA-PREP (or 800-426-8157).

Thank you for your support of our products

QIAGEN Inc.

-- 
QIAGEN       800-362-7737
Temporary Email Address          qiagen at kaiwan.com



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