Can i use same RE to check for insert? - blunt end cloning (Sep/16/2008 )
I had pcDNA 3.1 vector and I cloned in my gene of interest, checked the insert was there and in the right direction etc. I have also blunt end cloned in an EGFP reporter gene. I digested the pcDNA 3.1 vector with smaI and my EGFP insert with NruI (klenowed filled & cleaned up both etc) and then ligated the insert and vector (10:1) and transformed into XL Blue cells. All worked fine. I now want to check if my EGFP insert is in the colonies and in the right direction. I have miniprep'd some of the colonies and re-digested with nruI, the restriction enzyme i used for my insert. When I ran the digest on 1% agarose gel I got 2.4kb fragment (size of my insert) and a larger fragment (rest of vector I assume as pcDNA3.1 only has 1 nruI site) in a couple of the colonies. Does this mean my EGFP insert is there? I wasnt entirely sure if using nruI to check was the right thing to do.
Thanks in advance!
TCG | CGA
AGC | GCT
CCC | GGG
GGG | CCC
Both the SmaI and NruI sites are destroyed in the ligation reaction. NruI is not a very good diagnostic digest. I would suggest that you use some other enzyme to verify and orientate the insert.
It would be easier to choose a PCR primer on your insert and a primer on your vector and check presence and the size of the amplicon.
Great thanks!!! Will try both
Hi sorry to bother you again... i just looked over my protocol and realised I did it slightly different than i mentioned above.
I digested my pcDNA 3.1 vector with gene inserted with AflII and klenow filled. Then to take my EGFP insert from another vector I digested with SalI & klenow filled. I then digested this with NruI and gel isolated my 2.4kb fragment. This 2.4kb fragment was then ligated with the pcDNA3.1 vector i previously digested with SalI & transformed, miniprep'd etc...
Does this change anything? Do i still have to use a different enzyme/ primer to amplify?
If I am looking at this correctly you are ligation a Vector-AlfII klenow blunt ended to a SalI(klenow blunt end) - insert-NruI . This reaction should kill the NruI site. Do you have a plasmid manager to keep track of your plasmid construction such as VectorNTI?
I would suggest that you use some other enzyme.
Nope dont have plasmid manager to keep track of the construction. I'm going to look at sequence and see which restriction sites are upstream & downstream of my insert & try go with another enzyme! Will keep u posted! Thanks again!
I would strongly recommend your lab get one. It makes life a lot easier. VectorNTI is free. It can be tricky following all the restriction sites when you are building a plasmid.
Ok will definitely try download that! Thanks again!!