What PFGE device are you using and what are your settings?

Just a follow up in case I am busy later on.

In your case you want to have a better resolution of large fragments within the 19hour time window you have. I suggest you try using non-linear  switch time ramping where you ramp switch times from very short i.e. 5sec - 60sec for about 4-5 hours and use the remaining 14-15 hours to ramp switch times from 60-120sec. This should provide sufficient initial resolution of the smaller fragments while concentrating the remaining 15hours of the run on resolving the large fragments. I cannot promise that these specific settings will work but this is the only way you can increase resolution of the larger fragments without increasing run time. Also, looking at the distance of the shorter fragment bands a longer run time without proper ramping will result in the short fragments running to far. Give it a try and if the small fragments are still far apart reduce the 5-60sec ramping to only 3 hours and increase the 60-120 to 16 hours. If the large fragments are well separated but the smaller ones are not, increase the 5-60 to 5hour and decrease the 60-120sec ramping accordingly or try decreasing the initial switch time ramping to 5-45. If you need better resolution for both you might want to consider giving it a few more hours of run time.

Regards,

Manuel

Dear Manuel, 

Thank you for your suggestions.

We are using Biorad CHEF DR III.

We are trying the change in parameters considering your suggestions. 

Conditions/ settings used were:

o Initial switch time: 7 s

o Final switch time: 35 s

o Voltage: 6 V

o Included Angle: 120°

o Run time: 19 hours

Hi Godfred,

that explains the lack of resolution of the large fragments and the larger distance between the short fragments. In general you need short switch times for short fragments and large switch times for long fragments. You ramped rather short switch times which resulted in good resolution of the short fragments but you didn't include long switch times (60-120sec) to specifically resolve larger fragments. Try using my 2 block protocol and see if the larger fragments get resolved sufficiently well. You can also try a 3 block approach depending on the region you want to resolve. Again, short fragments require short switch times and large fragments require longer switch times. Let me know your result using my suggested 2 block protocol and we modify it depending on the result.

Dear Manuel NEB does not recommend to change the parameters/ programme for electrophoresis run.

Hence I have decided to go a head with the Biorad Lambda ladder, since the programme there matches my requirement for 19-20 hrs run.

Cheers!

I have tried all possible programme changes with NEB ladder but in vain. 

In fact I have earlier used Biorad lamda ladder with satisfactory results.

I wonder because NEB suggest ramping the switch time from 5-120sec. Can you post a picture of the 2 block protocol run?