Hi all,
I did ligation, transformation and minipreps, and then test digestion (EcoRI) to see if the inserts were successfully integrated in the plasmid. If the insertion were successful, I should see 2 bands 1.8kb and 8.6kb. If not, the empty plasmid also has one EcoRI site, should give a linearized 7 kb band.
However, the gel image showed bands all above 10kb, which confused me. Does anyone know why the results were like this? Thank you!
It looks like your original vector is not 7 kb as you report but larger in size possibly more than 10 kb. You might have isolated the wrong vector in the first place. Recheck the original source of your plasmid vector to confirm you have the correct vector. For confirmation of the plasmid, isolate the plasmid again from the correctly labelled original host cell and either check with some specific restriction digestion or sequencing of its characteristic and specific regions.
In your gel picture, it looks like you have a vector of more than 10 kb and your gene insertion/ligation has not worked. you have only got the self-ligated vector which after transformation and restriction digestion analysis shows the same linearized vector of more than 10 kb (the wrong vector). Your uncut gel image looks like the supercoiled vector and the EcoR1 digested image shows linearized vector with not insert.
Nothing unusual.
It happens many times when you are doing restriction digestion; most probably it is due to incomplete digestion.
You can do one or all of the following.
1. Reduce the plasmid concentration in the EcoRI digestion reaction to about 200ng or less.
2. Add at least 10 units of Eco RI in your digestion mix.
3. Incubate for 3h or more.
I bet you should get the desired result.
good luck.
Did you check the nucleic acid concentration? The bands are pretty thick in the non digested picture as well, which drives me to thinks that you samples are too concentrated.
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