perforated patch

 1: using amphotericin B, how long does it take normally to arrive at acceptable whole cell mode? I have used nystatin and I remeber it took several min.

10-15 min at least

2: Should I fill up amphotericin B in the electrode tipfirst, and then back fill the intra solution?

Backfill with a little normal pipette solution first at the tip and then backfill the remainder with the amphotericin containing solution.  This is so that when you are getting the seal you are not perfusing amphotericin over the whole cell and perforating the whole cell prep while you acquire the seal.  It will take longer to get stable a perforated seal but you can be confident that there is no amphotericin in the bath

3: Specific tips for handling amphotericin B. Stored frozen and once thawed, should it be used in a short (some min)?

Store in small aliquots
keep on ice in between patches
Sonicate the amphotericin solution for 30 s every time before you backfill a new electrode to ensure it is completely in solution.

Hi, i had a question about using nystatin . . .should my internal not have EGTA in it when I am recording using the perforated patch configuration.  And if its not, why? 

I use Amphotericin B for a long time. In my lab,  stock solution for Amphotericin B is made in DMSO(1 mg/40 vL, about one week use) which is stored in -20C .  about 5  vl of stock solution is diluted in 1 mL intrasolution immediately before use. Keep on ice, for a whole day use. I do not sonicate it between or before experiment.

What was the composition of your internal recording solution?  And did your cells swell and burst after you used AMPB/DMSO b/c I am having that problem

thanks