Resctriction analysis (View forum version)



Student30

Posted 09 March 2014 - 07:09 PM

Hello, I would like to get some help with something. I cloned a gen in pET TOPO vector 101 and when I did the analysis of the transformants with restriction enzymes(double digestion) I did not get what I was really expecting in the agarose gel. I just got one plasmid with the right bands that corresponded with the plasmid with the gen in the correct orientation, but the bands were very light. Furthermore, with the other plasmids I got bands that were not the bands I was expecting at all, not even if the gen was not in the correct orientation or the plasmid did not have the gen. I am not sure why I got thOse bands, I checked more than one time the bands I supposed I was going to get with those enzymes. Also, I am not sure either why the bands are as light. If someone could give some answer it would be very helpful. Thanks.

bob1

Posted 09 March 2014 - 10:50 PM

Faintness of bands could be that you didn't load enough DNA to detect those bands properly - the amount of DNA you need in each band depends on the size of the product in bp.

 

Multiple banding could be due to either incorrect restriction -either incomplete, star activity,or incorrect sequence or any combination of them.


Student30

Posted 16 March 2014 - 06:24 PM

Thanks for the answer!

 

Finally, It seems that there were two colonies mixed in the culture of LB I used to do the miniprep thus the bands in the restriction enzyme analysis corresponded with a clone with the vector in correct orientation and another clone with the empty vector.

 

About the amount of DNA I need what does it work?. If I have two gens one with 948bp and another one with 1100bp what is the amount of DNA I need?.

 

Thanks again


bob1

Posted 16 March 2014 - 06:50 PM

It works like this:  If you have a 10,000 bp (10 kbp) plasmid and you want to digest out a fragment of 1000 bp  the fragment will contain 1/10th of the total DNA digested (1,000/10,000).   So if you started with 100 ng of plasmid, only 10 ng would be in the 1000 bp band, which may not be enough to detect. 

 

So - long story short - I can't tell how much DNA you would need of total DNA as I don't know the initial size of the vector, but you would need about 5 ng of DNA in each fragment to ensure detection.


Student30

Posted 16 March 2014 - 08:04 PM

Ok, thanks! I understand now.


perneseblue

Posted 16 March 2014 - 08:04 PM

Light banding can be caused by having too little DNA or not enough staining.

 

As bob1 said, we need to know the actual size of the starting plasmid to give you a number for how much DNA to cut. For EtBr staining you generally need 100ng to see a band. But as a ball park value cutting 1ug of DNA is generally more than enough.

 

As for the incorrect banding patterns, the reasons are variable from incomplete digestion, to incorrect plasmid map, to having the wrong plasmid.

 

What you should focus on is your goal. Do you have the plasmid that you want? If not screen more clones. As my PhD supervisor was fond of saying, you only need one