Am doing an inverse PCR, and totally stuck in the digestion step. None of the enzymes I used seems to work at all ( BClI, MfeI or EcoRI).
I get a smear with the dense part only on the top, have tried lots of concentrations and digestion times but nothing is happening. I ran an un-digested DNA as a control and it worked fine though, any suggestion?
Thank you
Hi there,
What is strange is that you get the same negative result using three different enzymes... So it's either the three activities are gone (quite unlikely) or your DNA is not good enough for digest. What I would do is to check restriction enzyme activity on control plasmid and check the protocol for DNA preparation (there must be something wrong there).
The DNA samples were fairly pure once I extracted them, I also ran some of them on gel and looked just fine. what am thinking is that the elution buffer I used in the end might not be good or contaminated, or possibly have something that inhibits the digestion.
That's what I was pointing when writing about DNA preparation. Then if possible try to use new solutions. About elution buffer, try to avoid TE (10mM Tris-HCl pH8 is just perfect on its own)
What about the concentration of your purified DNA, and Quality of Digestion enzymes with positive and negative control. (Be sure to use suitable amount of both).
I really recommend you to try new purified DNA and you can use D.W instead of elution buffer in the final stage to dissolve DNA.
Also you have to remember that:
Too much DNA loaded onto a gel is a bad thing. The band appears to run fast (implying that it is smaller than it really is) and in extreme cases can mess up the electrical field for the other bands, making them appear the wrong size also.
Too little DNA is only a problem in that you will not be able to see the smallest bands because they are too faint.
Also you can upload your gel picture which contains undigested and single digested DNA. and Typical protocol which you have been used.
Good Luck
I am using the qiagen kit for dna extraction and the final step you use their elution buffer. i did re-extract dna again and will run on gel and see, if not working again maybe will use the dw.
once i get results will upload the photo. :)
thanks all.
Ok, it seems not working again, this time i used a 20 micro litre digestion reaction with less DNA, I used: 5 ul dna, 12 ul DW, 1 ul of enzyme, and 3-4 ul buffer.
left overnight in 37 degrees incubator, and ran on gel as: 2 ul of digested sample+.6 ul of dye loading buffer for 30 mints on 100 volts.
sadly, i can't upload ny photos here, now I guess the next step is to use dw as an elution in the last dna extraction step?
I uploaded a photo here.
http://s30.postimg.org/las3zhu6p/image.png
and this with undigested DNA as a control.
http://s27.postimg.org/cidg0kyjn/Ecor_I_digested_over_night_101114.png
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