I had found five bands after isolating PET28a vector by using a kit. I am using an EcoR1 enzyme for digesting PET28a vector but it is not digesting this vector. But along with this I had used PUC19 which can be digested easily. I am not able to understand this is happening.
I suggest you to check first if your purified plasmid is really the pET28a...maybe by digestion with pvu II.
Dear Varsha,
Hello. Did you find these five bands before enzymatic digestion? did you have any enzymatic digestion before the isolation?
Yours,
Heidar
You should end up with three bands before digesting a plasmid due to states of coiling. I would run uncut right next to a digest with an enzyme that cuts only once (say in your MCR) then you have a handle to base your next experiments on.
Morning Varsha,
I agree with Duncan's comments. If you are using EcoRI if should cut your plasmid once at position 193-192. The size of you linearized plasmid would be about 5300 bp.
I am not quite clear on what exactly you did, could you elaborate? Did you run a gel prior to and/or after isolating your plasmid? As Duncan suggested, run a sample of uncut and EcoRI digested plasmid at the same time. Secondly, if the "digested" plasmid is of a similar size as your circular plasmid, you may want to consider trying another restriction enzyme or re-evaluating the conditions you are using for your restriction digest.
Good luck.
Katherine
I suggest you to check first if your purified plasmid is really the pET28a...maybe by digestion with pvu II.
You have probably two different clones of bacteria that you used to prepare your plasmid. Basing on you description, you may have two different plasmids in your preparation.
You have to spread your bacterial suspension and screen for the right plasmid or you can make new transformation by the right plasmid before to proceed to the digestion.
Normally, using kit you find a plasmid preparation with 2 bands and maximum 3.
I think reason for 5 bands which you are getting its because of host DNA contamination along with plasmid DNA. You should check you kit reagents as well, if is there any contamination. Please do run uncut plasmid along with digested product and use multiple enzyme for digestion.
You might be experiencing incomplete digestion. if your sample contained only linear plasmid, there would be only one band. The fact that there are multiple bands suggests you have plasmid in different states (Supercoiled, nicked, linear, etc).
Here's an example: http://www.scienceforums.net/topic/53358-plasmid-migration-on-gel/
We've moved to 6 hour digests with high fidelity enzymes for pET28a in our lab in a thermocycler (37˚C for 6 hours, 85˚C for 20 minutes to denature the enzyme). NEB sells a HF version of EcoRI and we've had good success digesting up to 2µg of pET28a in 50-200µl reactions with 1µl of enzyme. All HF restriction enzymes from NEB need to be used in the correct buffer for their enzyme and adding 1/2 microliter of 100xBSA never hurts.
https://www.neb.com/products/r3101-ecori-hf
Good luck!
Iam getting five bands before digestion and after digestion i am getting two bands. One for linear and other is supercoiled form.Thank you so much for the suggestions.I think now i can solve this problem.
Can you post a gel picture? that would help a lot. After digestion two bands could be: linear (cut) plasmid DNA and supercoiled plasmid DNA (uncut); or contaminating chromosomal DNA (uncut) and the linearized plasmid (cut). If you still have supercoiled plasmid DNA after digestion then you may need to either increase the incubation time, or add more enzyme. Chromosomal DNA contamination can occur from incubating the cells too long in lysis buffer (
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