gfp digested with Ecor1 + Hind III, and Bgl II - (Sep/21/2006 )
I am new on molecular Biology and I need to know that the "GFP" plasmid that recive is really GFP. I have digested it with restriction enzymes EcoR1 + Hind III and Bgl II for to know the lenght of bands; somebody can help? or showing your pictures of the electroforesis gel after digest it.
Sorry for my english
Thanks so much
you can check this link
for an example of what cut plasmid DNA is supposed to look like (if the link is not good, go to 'google.com' and click on the 'image' search function; type in "plasmid DNA electrophoresis" and bunch of pictures will pop up)
do you have a map of your plasmid, showing enzyme cutting sites? from this map you should be able to figure out what size bands will result from your digestions. if you run a 'standard' or 'dna ladder' or something with DNA bands on known size, you can estimate the sizes of your bands and determine if your plasmid is what it's supposed to be.
also, it is good - even for experienced people - to run an uncut control if you are not sure of what you are going to see. this way you can compare it to your resulting bands and see if the digestion worked
[if u really want to confirm ur plasmid has GFP, then either sequence it or transfect it and look for fluorescence.
restriction digest could suggest GFP but not really confirm it.
The first thing you need to do is to find out what your plasmid is called. What is it's name? If a name can not be found the next thing you should do is to find out what variant of GFP is present within the plasmid. If a name can be found, look up the sequence at pubmed or a plasmid database. If you are unable to determine the variant of GFP, pick the most common variant used in most plasmids. Down load the sequnece from pubmed and indentify the GFP sequence. Find enzymes that will cut within the sequence. (VNTI is free for a year now. And NEB also provides a good enzyme cutter tool)
Does the digetion method work?
Alternative methods with increasing work
1- sequence the plasmid, particularly the GFP gene. Obtain the sequence and blast it. A blast search should return GFP sequences.... thus indicating presence of the gene
2-Find out if you GFP can be expressed in bacteria, e.coli. If yes, transform, tranluminate and watch the bacteria glow
you may pcr the CDS of GFP