Restriction digestion of PCR fragments

Richard Rohan rrohan at world.std.com
Mon Aug 21 21:25:46 EST 1995


pbv at rs.uovs.ac.za (B VISSER * x2818 Plantkunde *) wrote:

>Hi

>I am in the process of digesting amplified cDNA fragments with Not1 of which 
>a restriction site was incoporated into the primers.  I am not able to fully 
>digest the fragments, even though sufficient nucleotides are present on the 
>5' side of the restriction site.

How do you assay for cutting if the sites are near the ends?  How can you be
sure that the failure is at this step?

>Could Ethidium bromide play a role in the 
>inhibition of the digestion of the DNA?

This is possible since EtBr was used several years ago to create nicked or
single cut plasmid DNA by inhibiting subsequrnt restriction enzyme cutting
once the supercoils had been linearized by the first cut.

>I usually run the fragments on a 
>prep gel and cut the desired fragments from the gel with phenol/chloroform 
>which is followed by a ethanol precipitation.  Could this be the reason?

If you are worried about EtBr carryover, try a butanol extraction.  I don't
know if phenol/CHCL3 and EtOH precip will adequately remove EtBr.  I have
always used butanol extraction to do this.  BEWARE: Butanol partitions on the
top, aqueous on the bottom!  Opposite to phenol extractions.

It is more likely that phenol carryover from the extraction is the problem.
(Can be tested by measuring absorbance of final purification product at 260,
270 and 280 nanometers.  High A270 indicates phenol.)  Doing an extra CHCL3
only extraction after phenol/CHCl3 should help remove any phenol that has
partioned into the aqueous.  Also 70% ethanol rinse of pellet should help but
this step has a high probability of losing the pellet, particularly if you
only have small amount of DNA.

HTH


---
Rich Rohan, PhD (rrohan at world.std.com)
Independent Molecular Biology Consultant to the Internet
Moderator REPRENDO at world.std.com - Reproductive Endocrinology List




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