1) what is your DNA? (is it plasmid, or piece of genomic DNA?)

2) what is the fragment analysis?

Can you share picture of your gel? Did you run undigested control?

Dear Tomáš Hluska ,

Thanks for the clarifying question.

1. Piece of genomic DNA

2. Fragment analysis is a technique used to separate and identify DNA fragments based on their size. I am using this method to see the differences between microbial community in different soil type. My positive control is the ecoli that I used.

3. See attached Picture.

A. Bands (The third lane is the ecoli bands after digestion with RSA1 as per my reaction mixture)

B. My fragment analysis result of ecoli showing only one peak.

C. Fragment analysis result from a senior. Ecoli showed multiple peaks.

Tomáš Hluska, sorry here is another photo of fragment analysis of ecoli by a senior that showed multiple peaks.

Do note, that my research fragment analysis was done in Malysia and another one in australia. We had different provider.

I was just wondering by the look of my gel band if my restriction digestion was successful. But then, how is it that my fragment analysis result showed only one peak, and not multiple like my senior.

If you are familiar with this protocol, do share your thoughts.

Thanks Tomas.

How many bands did you expect? If you know the sequence, then you should be able to predict your results.

A proper positive control would be a piece of DNA that you know exactly how it will be cut by your enzyme. And also run some non-digested DNA from your E. coli as a comparison.

Good luck!

Thanks Katie A S Burnette , unfortunately I don't know the sequence. Would you recommend sending the DNA for sequencing first before I proceed to run digestion? So that I can be sure of the cut site.

Wait, you don't know the sequence for your positive control? Why are you doing a restriction digest?

Yes, I would recommend getting the genome of your positive control sequenced first so you can design your assay.

are ypu amplifying dna with a dye labelled primer in which case only one cut fragment can have the dye incorporated so the agarose may show multiple bands but only one end digested fragment ( and any uncut amplimer) will show on fragment analysis which reads the colour of the dye but any other unlabelled fragments will not be visible. If the dna is a mixture of different species then different sixe labelled fragments may be produced depending on where the first cutsite is

Katie A S Burnette, Yes I didn't know my sequence. I was following a protocol from a senior that previously have done it. But ever since I felt that something is off somewhere and needed to be checked, I decided to dig deeper and seek advice from those who are familiar with this process. Now I find it makes total sense to first know the sequence before running digestion, or else it is like shooting in the dark.

And thank you for your kind advice, it has now given me multiple solution to solving my problem. Greatly appreciate it!

Inheriting a plasmid project is basically a guarantee that something is going to be not as expected. yes, sequence it & you'll be able to solve your digest problems.

Good luck!