| 1) When making intracellular solution how careful should you be in measuring out salts, some like EGTA are in such tiny amounts that you cant possibly weigh them accurately even for a large volume like 100 ml. |
You should always be
very careful - a way to accurately add small amounts of any salt to your intracellular solution is to have a concentrated stock of solely that salt which you then pipette into your mixture using a calibrated pipetteman.
2) I left my intracellular solution at room temp for 2 days. It has a drug (QX314) in it, should it be ok, or I need to trash it? |
Call the supplier or check the MSDS for stability properties in solution. However, If you have plenty of the compound it would just be better to make up fresh drug to be safe. Also aside from your dug, many intracellular solutions contain ATP. If you have ATP in your solution you should keep it on ice even while you are between patches. If it is left thawed for longer than a few hours at room temp I don't expect it to be good.
| 3) is it ok to use millipore water or do i need to use RNAse and DNase free water. |
Millipore is OK - just pass your final solution (after pH-ing) through a 0.22 um filter before you use it.
| 4) What is the best way to adjust pH. I have noticed that pH changes substantially with increasing volume. I make 140mM CsCl solution. The pH is usually was way off 7.3. I was using ~300 mM CsOH to adjust it, and ended up adding a good volume. Maybe ~4-5 ml. Is it ok to have a 5-10% error margin in volume, if the osmolarity is in the right range? |
Plan ahead - try and make up your solution in 50-75% the final desired volume. pH that and then increase to the final volume monitoring the pH as you go. Use more concentrated CsOH -e.g. 1M so you add less volume to increase the pH to 7.3
| 5) Is a 270 mOsm osmolarity ok? |
270 mOsm is maybe a little low but probably OK; but ask yourself this. What was the osmolarity of the culture medium in which the cells your are recording from were grown? Typically culture media comes out at about 296 mOsm. It's best to try and match your solutions to the osmolarity of the culture media so there is little osmotic shock before you patch. For a good seal, the practice is often to make your Pipette solution have 5-10% lower osmolarity than your extracellular solution so that the cell swells slightly when patched by the pipette tip making a better seal.
Common solution combinations include 296 mOsm Internal/325 mOsm ECF, or 280mOsm Internal/ 296 mOsm ECF.
| There are so many of these small questions. is there a good practical guide that beginner's like me can refer to? |
I usually recommend "
Patch Clamping" by Areles Molleman as an easy beginners guide to patching
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