HELP in Restriction Enzyme Digestion - troubleshooting re digestion (Sep/25/2007 )

hi. i am student currently working on a project titled sexing of monomorphic birds.

the sex gene of birds are ard 110bp.

im now at the last stage which is cutting the DNA with restriction enzyme DdeI to check for the gender of the birds.

im using the following formulation:

10X NE Buffer- 1ul
DdeI- 2ul
PCR Pdt- 5ul
Distilled water- 2ul
Total volume- 10ul

I have tried for several times but it the enzyme does not cut the band. i have tried incubation for 16hrs, 24hrs, even 4 days at 37degree celcius. but most of the time it doesnt not show any band. not even the original un-cut band!

recently i tried this formulation:

10X NE Buffer- 2ul
DdeI- 1ul
PCR Pdt- 15ul
Distilled water- 2ul
Total volume- 20ul

This time the 110bp uncut band is showing but nt the cut bands.

Is there smth wrong with my restriction enzyme? i read on some forum that BSA can be use to help protect enzyme, should i use BSA with DdeI?

Or is the cut bands of 80bp and 30bp to small to be detected on the gel? i use 2% agarose to run the gel, usually run at 110V for ard 40mins. What can i do to get results? pls help. thank u :]

I would try to clone your PCR product in something like a topovector (Invitrogen) before doing the digestion. In a vector, you will have some bigger fragments to look at. Anything smaller than 100 is quite difficult to see on an agarose gel. You could also try to use an acrylic amid gel instead.

Good luck
/Marianne

I agree with Marianne, but you also should use your second formulation, because an enzyme volume higher than 10% of the total volume may result in inhibition of the reaction due to the glycerol that is in the enzyme mix. The glycerol content never should be higher than 5% in the final volume.

Hi,

Have you tried to cut for shorter times? I would suggest a volume of 30 ul final, and a 1-2 hour digestions.

As for seeing the bands, well, if you can see the 100bp there's a good chance you would see the 80bp too. Not sure about the 30bp, though.

Good luck!

Alexandre