Experiment detail:
I am digesting the pCMV6 Neo Vector of 5838bp and pCMV XL5 of 4700bp with newly bought Not1 enzyme from Promega in separate reactions for 2h at 37 C followed by heat inactivation at 65C for 15 min.
After digestion, I purified with phenol:chloroform:isoamylalcohol (25:24:1) (Sigma)
All of them were analysed in 0.7% gel against 1kb ladder from Promega.
Observations:
The digested products were showing higher than expected i.e
i, the pCMV6 Neo instead of showing around 6kb shows up at 10kb
ii, the pCMV6 XL5 instead of showing around 5kb shows above 8 kb.
iii, the purified product as described above shows multiple bands.
This observation is very consistent in repeated experiments with the same conditions and also in modified conditions like using 4-5 enzymes units per ug of DNA.
I suspected the marker could be a problem, hence verified with a known pcr product of 5kb which exactly corresponded to the marker lanes we use in the lab (data not shown).
I also suspected that the enzyme could be still bound which made my digests running higher, hence I added SDS to 0.5%, 1%, 2% but I didnt see any shift in size.
Below I attached the gel picture.
My questions:
1. What could be the reason for seeing the higher size band, and how can I overcome this?
2. Do the multiple bands after PCI extraction give any clue to the digestion or is it just the artifact in my purification itself?
Kindly give your suggestions to help me move forward.
First of all it would be wise to include the original, uncut product to a gel. It wouldnt surprice me to run at the same heigth as the 'cut' productucs.
What is the pattern you find from other restriction reactions? Do they work properly?
The PhCl purified product may contain a little DNA of the right size. The big lower band is a mistery to me to, or does the vector have two NotI sites?
Something similar happened to be a while back. I ended up retransforming my plasmid DNA into new competent cells. For some reason, the DNA from my original preps was not what i expected. It was an issue with my competent cells. I suggest you make new DNA preps and try again.
Hi
I would also load the uncut next to the NotI treated and to see the size of the linearised fragment cut it with another enzyme too. If possible a single cutter, otherwise something that leaves a big fragment.
And you have terribly smiley bands, maybe load a bit less or run your gel slower (especially the first 5 min).
@Aurelie, some hosts methylate restriction sites and make it hard to digest, this can be circumvented with retransforming it to another host that does not methylate that specific site. I don't think that is an issue woith NotI though
Hi
NotI restriction sites are not methylation sensitive. I suggest you incubate your digestion mixture with NotI enzyme at 37 degrees for 16 hours. Also you may run the uncut vectors nearby the cut ones. Moreover for NotI enzyme inactivation, you may incubate it at 80 degrees for 30 min.
In the case of extra bands in the lane 4, you may check the restriction maps of used vectors. they very probably have more than one NotI sites. If you want to recover the linearized vectors from gel, after inactivation of restriction enzymes, no needs to PCI purification.
God speed you
Hi, great that you have uploaded a gel picture. I may repeat previous suggestions, sorry about that.
I would suggest you run the gel longer and/or use a larger gel chamber if at hand. Separation might be essential here, as the fragments of interests are really close on your picture.
Moreover, the ladder is quite "smiley" which makes the determination of sizes different. I remember faintly, that I once had a ladder in which the highest part of the smile corresponded best to the lowest part of the linear DNA on a gel. Anyhow, I cannot explain the basis for this observation and this might be crazy-talk. Again, long separation on a gel is important.
For the additional bands I can only speculate. Maybe you should load undigested DNA as well (ladder, undigested, cut, cut and PCI purified). Previously, I had one additional band at a lower size, which the turned out to be ssDNA due to excessive alkaline lysis. Surprisingly, I enriched these DNA species more efficient than others, I thought. A new DNA prep (as suggested by others) with less alkaline lysis or column purification might help.
Hi
It was happened with me previously with NEB Not I enzyme.
I suggest you to do fresh DNA prep and digest pCMV vectors(200ng) in 20-40uL (keep lower conc. of your DNA and enzyme). Then run 1% agarose gel for long-run with uncut DNA.
1, What could be the reason to see the higher size band, how to overcome this?..Maybe u used VERY high amount of DNA or Your agarose gel time is to long for it.
If all else fails, sequence your vectors to make sure they are what you think they are. Things aren't always as they seem, especially if they were prepared by someone else.
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