I am trying to clone a gene in expression vector pET 32a but while doing so when I run a gel (0.8 agarose) with cut and uncut vector I got some weird result. The cut vector resulted in band of 4 kb approx instead of 5.9 kb. What could be the possible reason for this? I have use bam HI as restriction endonuclease since it have one one site in the vector, so ideally vector should give linear fragment of 6 kb aprox.
From what you describe, my guess is that the DNA you have is not pET 32a. Is the source of the plasmid DNA reliable? I would first try to cut the vector with and enzyme that cuts more than once so that you can see a recognizable pattern. If the restriction fails again, then that DNA is not the plasmid you think.
Is there difference in the mobility of cut and uncut vector? How many microliters from the restriction reaction you load on the gel? Because sometimes the salts from the buffer could make the DNA to migrate anomalously if bigger volume is loaded. Check the restrictase on another vector too, to be sure firstly if the enzyme is working ...
Thank you for you valuable suggestion. I will try both approach. As per the information i had used 5ul plasmid of 396ng/ul conc. and did the restriction digestion up to final volume of 10 ul and loaded all in a single well. I have attached the pic also. Left hand side with respect to marker is uncut pET 32a while right hand side is the cut one.
Hi Kashif,
In the uncut lane you have circular and circular supercoiled (that generally runs faster than its expected size) in your cut lane you have mainly linear that will run similar to circular (not supercoiled) so I would say the enzyme is working. I cannot comment on the sizes as I don't know the marker sizes.
With regards to size-do you have EtBr or SYBRSafe in the gel? These can both make bands run anomalously-try staining after the run. Also 2µg in a single lane is a lot-this can also affect the band migration-try loading the equivalent to a single marker band (probably 50-100ng?) and see if it runs correctly.
If it still runs at the wrong size after this then question the source/ID of your plasmid.
Dear Nick,
I have used EtBr incorporated into the gel, and DNA ladder is of 1kb in size which mean the lowest band is of 1 kb followed by 2 kb till 8 kb correspond to 8th band and the last and topmost band of 10 kb i.e. 9th band. Thanks for your suggestions of staning after run i will definitly try that.
I think it's worth trying a control digest with a different restriction enzyme with a smaller amount of vector; I second the idea of a double cutter or a double digest (like EcoRI and PstI). The size of the cut band on the right looks indistinguishable from the larger uncut band on the left, and there is too much DNA in that lane to make a good determination of size of the band.
BamHI RE has star activity.
To confirm the vector try and use other restriction enzymes like HindIII, EcoRV or XhoI
Kashif,
Given that your pET30 digest is only running with one fragment smaller than expected, it is quite possible that your plasmid does not contain your gene of interest. Digest with a different enzyme that has a site within your gene of interest and run the gel again. You preferrably never want to run a gel with a digest that will produce only one band. Find an enzyme with 2 or 3 cut sites (one being within your gene).
I see nothing wrong with your digest. You have killed most of the supercoiled band by cutting, so the enzyme is working, and as far as I can see your major product is the right size. From your description of the ladder I don't know where your 4K number comes from.
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