very high molecular weight plasmid - (Apr/28/2010 )
hi everyone. i recently prepared maxiprep of a plasmid with gfp. this plasmid was commercially available and just transformed them and prepared maxiprep. the plasmid i purified has an OD of 1.9 so i think its okay however when i checked it in agarose, the size is very high, higher than the first line of the dna marker. I expect that the MW of my plasmid would be around 5-6Kb only.
Did you digest your vector before running it on a gel?
Non-linearised DNA will run slower than linear DNA and hence look bigger in size compared to a DNA ladder.
soymilk14 on Apr 28 2010, 02:57 PM said:
hi clare, no, i did not digest it. i can try digesting it then clare and check. it is a plasmid bought commercially, harboring a gene fused with gfp. it was running out so i transformed it and prepared maxi prep. i just dont know why after running its molecular weight is very high. could it be chromosomal dna?
How did you purify the plasmid? If the DNA is running near the wells, it could be chromosomal DNA contamination.
You can not get an estimate of a circular DNA's size by comparing it to a linear DNA ladder...
HomeBrew on Apr 28 2010, 09:03 AM said:
oh thank you thank you. i purified it by etbr-phenol-chloroform after maxiprep. are there any suggestion how to confirm if my plasmid i run on the gel and purified is correct. sequencing?
Sequencing is one way. Another less exact method is to digest the plasmid with a restriction enzyme that linearizes it, and then you *can* get an estimate of its size compared to your ladder.