What is wrong with my EcoRI digestion? - Can it attribute to the star activity? (Feb/28/2007 )

Yesterday I did a EcoRI digestion of recombinate pcDNA3.1, which is 5.5 kb containing a 1.5 kb insertion.
According to the restriction positions, I should get two fragments : 5.5kb (the linearized pcDNA3.1) and 1.5 kb insertion.
However, I got a very light 750 bp band and a stronge 5.5 kb band.
My reaction system is as the following:
Multipore 10X Buffer: 2.0ul
BSA 0.2ul
Water : add to 20 ul
Plasmid DNA: (total amount= 2 ug)
EcoRI 1.0 ul (concentration: 12u/ ul, 1.0 ul=12 u)
Total volume----20 ul
Incubate in PCR in 37℃ for 4 hours and then 4℃.
Electrophoresis: 1% agarose; 10 ul reaction product plus 2.5 ul loading buffer.
I have uploaded the gel image.
Thank you for your help!

-sunnyhut-

QUOTE (sunnyhut @ Feb 28 2007, 11:05 PM)

I can`t see your gel image. But as I know, pcDNA3.1 (Invitrogen) is 5.0Kb. I suggest you check your insertion`s RE site.

-Hyland-

have you sequenced the insert? perhaps there is a site that you didn't know about somewhere in the middle

also, 4 hours is a very long time for digestion with EcoRI; it may be star activity

it would be best if we could see the pics, though!

good luck

-aimikins-

the amount of enzyme for the quantity of plasmid used is quite high. I recommend either increasing the volume of the digest (~50ul) or using 0.5ul of EcoRI.

-perneseblue-

QUOTE (perneseblue @ Mar 1 2007, 02:23 PM)

the amount of enzyme for the quantity of plasmid used is quite high. I recommend either increasing the volume of the digest (~50ul) or using 0.5ul of EcoRI.

not able to see the pics.

start activity is possible for ECOR 1.

try digesting in 50ul volume and digest for 1 hr. its usually sufficient.

-scolix-

Now I know the reason. The plasmid that I got from other people is empty one (without insertion).
Yesterday , I just got the exact result as expected, both in EcoRI and EcoRI plus BamHI digestion.

Thank you all!!

-sunnyhut-

-perneseblue-