I got 2 bands after restriction digestion of unknown gene fragment, does it mean that my gene has restriction site apart from vector. Can I assume that addition of these two band will give me the actual length of my gene fragment....what is the other method to identify the restriction site in gene fragment, ladder starts from 250 bp and vector is PTZ R/T. The gel pic is attached below, first lane is ladder, second lane is uncut plasmid, third lane is restriction digestion product. Please give your suggestion....
Hello Qurshid,
It seems that you have restricted a vector that contains your gene of interest. You got three bands. You mentioned 'unknown gene fragment'. If it is 'unknown' then how are you assuming that restriction enzyme is cutting within your gene of interest? Would you please rephrase your question?
Yes, it is likely that your unknown gene fragment contains an additional restriction site. In this case it is possible to add up the fragment sizes to get the complete size of the plasmid and thus the size of the insert. An alternative method to check the plasmid is PCR, preferably with oligonucleotides flanking the region of the insert. Sequencing of the PCR product gives you the information you require.
Cheers, Christian
Hi Syed Ali...
In that three bands, first one is plasmid (PTZ R/T) and the two fragments I assumed as my gene of interest, as it is an unknown gene that is why I thought that there will be restricted site resulting in double band....
A gene is cloned in pTZ R/T vector. Why it is unknown? This is what I am wondering about? Where this gene came from? The pTZ R/T is a T/A cloning vector. So the gene cloned in this vector must have been PCR amplified. If it was PCR amplified, sequence information was available (after all primers were designed based on sequencing information).
So you have chosen a restriction enzyme that cuts once inside the vector. Because you are getting additional bands, you are assuming that it is also cutting inside the cloned DNA fragment. Yes, it looks like this.
I am doing RACE to identify the whole length of the gene. I have partial fragment and by utilizing that I am synthesizing the full length. The full length sequence pertaining to that specific gene is not available in database.
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