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Pulsed Field Gel Electrophoresis

PFGE smearing no bands

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#1 theofilospapad

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Posted 16 February 2012 - 10:48 AM

Hi,

sorry for my English...

I have a really big problem with PFGE, we use a Biorad chef mapper3

we tried to run a low range pfge ladder (neb) and a lamda ladder (neb) but the result is on this image...

the running buffer is 0.5 TBE.

Thnx for your help!1s.JPG

#2 phage434

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Posted 16 February 2012 - 11:04 AM

What temperature? What pulse switching rate? What voltage? How long?
I don't understand why people expect us to guess these critical parameters when they ask for help.

#3 theofilospapad

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Posted 16 February 2012 - 11:41 AM

2,2sec - 63.8 sec run time 19h temprature 140c , 6volts. All the parametres were from the cdc-pulsnet protocoll for salmonella

Sorry...but I thought it is not important. Thnx again

#4 phage434

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Posted 16 February 2012 - 05:46 PM

I have found that the lambda ladder has poor resolution when run with fragments much longer. I'd recommend trying the lambda ladder with a run optimized for 20-200 Kb fragments, rather than one including megabase fragments. I've never had good luck with the mid and low range ladders, and much prefer the yeast chromosome ladder. If you are aiming for small size fragments, you definitely don't want to have such long switch intervals. The total length of your run also matters, longer is better.

As a comparison point, look at the gel running conditions for the NEB low range PFG ladder. 6 v/cm, 1% gel, 15C, but switching times from 1-12 seconds, run for 15 hours.

http://www.neb.com/n...roductN0350.asp

#5 theofilospapad

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Posted 17 February 2012 - 01:57 AM

Thnx for your answer


I suppose the problem is on chef mapper, I have runned the gel several times in 2011 with very good images with 2,2-63,8 etc. I checked agarose, TBE buffer and seems they are ok.

Here you can see an image from 11/2011. the first and the last lanes are low range ladder from NEB, the same I use every time.

Possibly I must contact Biorad- service

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  • 2.jpg






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