[DIYbio] Re: Pulse-Field Gel Electrophoresis - problems with the DNA damage

What type of DNA damage are you trying to look at? How are you purifying your DNA?
Smearing on gels is usually caused by nucleases or DNA shearing from misshandling.
Need more info on what type of protocol you are using if you want more help.


On Thursday, October 4, 2012 9:21:49 AM UTC-5, szydel wrote:

Hey guys,

I am working on the DNA damage on Adineta ricciae bdelloid rotifers upon the desiccation. I'm resolving chromosomes on CHEF DR III system (BioRad). The probem is that I see the DNA damage even in wet samples. The only reference I found on the literature is about Adineta vaga from Gladyshev and Meselson (2008) paper ( http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2278216&tool=pmcentrez&rendertype=abstract ). Do you have any experience in PFGE? The conditions I used in my runs were:

14-09[2]: 22 h at 14oC, 50-90 s switch times, 6 V/cm 120o angle in 0.5x TBE buffer

20-9[long2]: 72 h in 3 blocks:
Block 1: 14°C, 20 min switch time, 2 V/cm, 96° for 24 h in 1xTAE buffer
Block 2: 14°C, 25 min switch time, 2 V/cm, 100° for 24 h in 1xTAE buffer
Block 3: 14°C, 30 min switch time, 2 V/cm, 106° for 24 h in 1xTAE buffer


Many thanks,

Luke

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