Whole Human Genome Restriction with Mfl I

[incub0x]

Hi:

I am a PhD student, I am trying to get Whole Human Genome Restriction with Mfl I. My conditions are:

10x L Buffer-------2 µl
DNAg ---------------1 microgr
Water deionized----up 20 µl
Mfl I-----------------1 µl (10u/µl)

37º C--------18 hrs
65º C--------15 minutes

But I have not obtained restriction of the genome. For that reason I am consulting you about this. Maybe I am doing something wrong or I need to do something more.


I appreciate your advices.


Thank you.

 

[CharonY]

I presume you are using ultrapure water (or double-distilled) not merely deionized? More importantly, what % is the gel and is it really a 100bp ladder (a 6-mer enzyme should yield fragments with an average size of about 4 kb...

[incub0x]

I am using milliQ water. It is an 1.5% agarose gel and Is is a 100bp ladder

[CharonY]

In that case, the restriction looks pretty much as expected. It is just not resolved in the high MW area (the top would be around 1 kb). To see more (and see if it worked) you should go with 0.5-0.8%.

Edited July 14, 2014 by CharonY

[incub0x]

I appreciate your comment, I will try, thank you very much