I used two restriction enzymes to cut my PCB1532 plasmid but I do not know when I run on gel how can I distinguish if my enzyme are not work properly. I cut this vector to use on fungal complementation.
Regards
Ayat
Generally linear(i.e, cut) plasmid go with the size of plasmid which you can read using DNA ladder. If you are digesting ur plasmid with two enzymes you expect an insert release, say only one of ur enzymes is working then u see only linear product. Loading uncut plasmid would be a proper control in both the cases to differentiate.
Hello,
Ayat,
Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. As Paul lesbats suggested u can run the plasmid cut and uncut with ladder.. uncut plasmid will appear on the bottom of gel while linear will appear top of the gel.
Good luck...
Hi Ayat - a good answer from Paul. Did you use two different enzymes? If so you ought to get two bands from the digestion, not just one (provided of course that they don't cut your plasmid in half!). However, the key, as stated, is to run the cut and uncut in parallel.
Remember that "uncut" in this context is:
- equal amount of plasmid,
- in the same buffer,
- adjusted to the same volume to account for whether Enzyme is present or not,
- incubated at the same temperature for the same time,
- and the same volume of this mix ("digestion" or "mock digestion") added to your losing buffer for the gel.
Make a 0.5 or 1% agarose gel and load:
X X L X X C X X U X X L X X
where:
X = empty well
L = Ladder
C = Cut
U = Uncut
You may wish to supplement this experiment by using each enzyme individually as well as both together
Linearised plasmid will appear heavier than coiled plasmid and double digested plasmid will appear smaller than linearized.
Good Luck
G
Thank you very much for all. It is good and helpful answers. Actually, I used 2 restriction enzymes to linear PCB1532 plasmid. I used ApaI and SpeI to cut this vector, the fragments which I should get from cut 70bp. PCB1532 vector size is 5233bp if I cut just 70bp it is difficult to recognize by the gel and measure by ladder.
Hi Ayat,
For small DNA size, you can prepare higher agarose gel concentration and run at lower voltage. Higher gel concentration (up to 4-5%) will result in better DNA separation for a small DNA size. You also can use small DNA mass ladder such as GeneRuler Ultra Low Range DNA Ladder. Good luck
Hi, Usually the plasmids are circular shape and appears around the middle of the gel system and the linear one appears at the bottom. as linear can move easily through the gel it goes down and the circular one at the middle due to less mobility through the gel.
Be careful with chloramphenicol since it can change the way plasmid migrates.
I want to linearise PCB1532 vectors. My vector without insert
Hi Ayat.
OK. Vectors are by nature circular and, because of the helical nature of DNA can resemble a knot. To give yourself an idea, take an elastic band, hold one end and twist the other.. see how it knots up the two ends move closer, and so it appears to get smaller? Now, imagine that your isolated plasmid will be in various stages of this coiled state.
Compare this with a same-size elastic band that you have cut once with scissors... lay them out along the side of a ruler.. the cut plasmid (ie elastic band) is "longer" than the uncut one, which *appears* shorter on the ruler, even though it is the same length.
Hold that image!
Now consider the gel. Gels are like three dimensional nets. We talk about things migrating through gels as if it was something they did by themselves, but in reality they are pulled through by the electrical potential across the gel.
With any %(w/v) agarose, the distance between the "strings" (cross-links) that make up the "net" (agarose gel) varies, and the higher % agarose, the closer the strings and so, the smaller the holes.
To move through the gel, your cut plasmid has to be pulled through the holes. A smaller object gets pulled through more easily than a large one and so, your linearised plasmid will not migrate as far into the gel as the non-linearised one. Consequently - linearised plasmid appears *heavier* on an agarose gel than circular plasmid (think of the elastic band image).
Linearising the plasmid requires a *single* cut, not a double one. Any enzyme that cuts the plasmid once only will linearise it. Each of the enzymes you mention will produce linearised plasmid appearing to be the same size. If you use both then the 70bp fragment will be out of the gel and will not be visible.
Your plasmid is 5.2kb - this is easily resolvable on a 0.5% agarose gel using a 10kb ladder to calibrate.
Finally, think about your loading dye - bromophenol blue will run very small on 0.5%; you may wish to try xylene cyanol instead (nicer colour, too!)
Good luck
G
Thank you very much Grant Gallagher for this explaination. After I read your nice example, I will remember it forever.
Thanks for all people who sharing their idea. It is so helpful
Just one quick question to add.. In linearizing a plasmid to use as a qPCR template for absolute quantification (of e.g. gene copy number), would it matter how many times the plasmid was cut, as long as all of the cuts were outside the insert?
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