6 replies to this topic

[soymilk14]

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.

[Clare]

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.

Clare

soymilk14, on Apr 28 2010, 02:57 PM, said:

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.

[soymilk14]

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?

[epibio]

How did you purify the plasmid? If the DNA is running near the wells, it could be chromosomal DNA contamination.

[HomeBrew]

You can not get an estimate of a circular DNA's size by comparing it to a linear DNA ladder...

[soymilk14]

HomeBrew, on Apr 28 2010, 09:03 AM, said:

You can not get an estimate of a circular DNA's size by comparing it to a linear DNA ladder...

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?

[HomeBrew]

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.