NEED HELP-verification of my inserts - (Aug/29/2007 )
Hello everybody,
I have an expression vector about 4600 bp. I cloned my insert in it and made colony PCR. I got very good amplification, so I isolated the plasmids and digested if everything is okey. It should give one band around 360 bp, but now I have 3 bands which are 250,360 and 650. Any idea?
do you have and, if so, what are the positive and negative controls for each of these steps ?
First, my insert is PCR product. After purified it, I have cloned it into pJET1 plasmid and verified it by sequencing. Then, cut and run in agarose gel. Everything was okey. Then, I cloned it into expression vector and choosed the colonies with proper antibiotics. It cannot have any parts from plasmid now, because pJET cannot survive where my expression vector lives. From positive colonies, I carried out colony PCR, it gives good amplification and the band level is okey. Therefore, I isolated it and cut if it is ok. Unfortunately, I have additional bands. I have also control for digestion. Enzymes, buffers and vector work well, there is no problem. Any idea?
is there a possibility of concatemerization ?
It could be that your plasmid has sites for one of the enzymes you're using.. I assume you checked that, though, so I wouldn't bet on it...
It could be concatemerization, but I'm not really sure, since it would give out the same band wieght and you would not note it.
I'm running out of options, and in my lab my working partner has the same problem, she should get one band at 2.2Kb, but she gets also one at 1500...
You could perform a single digestion to see wich enzyme is cutting more than once and then see if that brings you more ideas... If it does, post them here so we can read them 
If we get any idea with our own problem, I'll post it here 
I have an expression vector about 4600 bp. I cloned my insert in it and made colony PCR. I got very good amplification, so I isolated the plasmids and digested if everything is okey. It should give one band around 360 bp, but now I have 3 bands which are 250,360 and 650. Any idea?
IF PCR gives you a single amplicon product at 360 bp, you have a good clone/good clones. Be very happy.
The fact that RE digestion gives you a number of products means that there are extra sites. Not happy.
If you want to move the insert from one plasmid to another, can you not simply do a PCR from the purified plasmids and clone into the new plasmid? On the other hand, if you want to express what you have cloned, you could simply do 10 or 20 small-scale (2 ml in a 10 or 15 ml tube) mini-expression experiments. Pick separate colonies, grow for 2 hrs, induce for 2 hours, spin down the cells, lyse them and run SDS-PAGE. Once you have clones that express, you can worry about levels of expression, soluble/insoluble etc. The whole expt is over in a day.
There is no law against being very pragmatic!
I have an expression vector about 4600 bp. I cloned my insert in it and made colony PCR. I got very good amplification, so I isolated the plasmids and digested if everything is okey. It should give one band around 360 bp, but now I have 3 bands which are 250,360 and 650. Any idea?
IF PCR gives you a single amplicon product at 360 bp, you have a good clone/good clones. Be very happy.
The fact that RE digestion gives you a number of products means that there are extra sites. Not happy.
If you want to move the insert from one plasmid to another, can you not simply do a PCR from the purified plasmids and clone into the new plasmid? On the other hand, if you want to express what you have cloned, you could simply do 10 or 20 small-scale (2 ml in a 10 or 15 ml tube) mini-expression experiments. Pick separate colonies, grow for 2 hrs, induce for 2 hours, spin down the cells, lyse them and run SDS-PAGE. Once you have clones that express, you can worry about levels of expression, soluble/insoluble etc. The whole expt is over in a day.
There is no law against being very pragmatic!
Yeah, it seems that it gives single amplicon, but as I mentioned before RE digestions are terrible. I couldn't find extra sites, but yesterday I realized that if my insert+the small part after digestion of expression vector are cloned in the same vector, digestions can give that much bands.
Fortunately, I have 3 different colonies and one gave the best digestion. So, I can keep on cloning my other insert.
Thanks for your help..
It could be concatemerization, but I'm not really sure, since it would give out the same band wieght and you would not note it.
I'm running out of options, and in my lab my working partner has the same problem, she should get one band at 2.2Kb, but she gets also one at 1500...
You could perform a single digestion to see wich enzyme is cutting more than once and then see if that brings you more ideas... If it does, post them here so we can read them
If we get any idea with our own problem, I'll post it here
Thanks for all your help, but as I mentioned before my vector doesn't have the sites for these enzymes. I have checked several times. If you have any additional result from your studies, I will be glad to hear them. I have overcome this problem by changing my clone, but I still have questions on my mind.
might be a stupid question but:
after cutting out of pJET1 did you run a preparative gel and cut out your insert and did gel cleanup to be sure just to clone the insert into expression vector
and not also some by-products from the restriction?
can i know wat enzyme u used... how many colonies u get.. whether all colonies got same result after restriction digestion