Lysis Buffer: Help Deciphering Protocol

[sobored]

lysis buffer- help!

the protocol for the lysis buffer i use is:

5mM Tris
1mM EDTA pH7.4

does it mean that the lysis buffer has to be pH7.4 or does it mean that the EDTA buffer alone is ph7.4? can you guys tell me which one of these options is correct?

1. add EDTA and Tris to the same bottle with the correct amounts and adjust the pH to 7.4. the lysis buffer will be pH7.4


2. make a stock solution of EDTA pH7.4 and add the right volume to the Tris solution. the lysis buffer won't be pH7.4 (due to the Tris)

i appreciate for any ideas!
thanks alot!

 

[chem_tr]

Well, I appreciate your tries to fully understand the process. You are doing the correct one.

As you know, Tris is a buffer with changeable pH values. If you add some EDTA (disodium salt, I suppose), the pH value slightly shifts to the alkaline side. It is possible that they noted the final pH of the lysis buffer.

Maybe you need to measure the plain pH values of both of the solutions you are using. I think Na₂EDTA has a pH near 7,4; and tris buffer about it. Then you will devise a strategy.

Since it is a pH-dependent biological-material process, I recommend that you first learn the correct option by measuring the starting pH of the solutions.

Regards
chem_tr

 

[sobored]

You are doing the correct one.

thanks, for your reply! but which options do you mean? is that 1 or 2?

 

[Monique]

I would make 5 mM Tris, adjust it to pH 7.4. Then add EDTA to 1 mM. Since Tris is a buffer: addition of EDTA shouldn't change the pH, I would just write down into your notebook how you're making it and what the pH change was before and after addition of EDTA.

 

[sobored]

Thanks monique! but where can i find my notebook? i am new here.

 

[Monique]

Always write down into your lab-notebook exactly what you are making and how. If something does not work as planned, you can always look back and see what went wrong.

 

[sobored]

okey! so you meant my lab- notebook? understood.

 

[Monique]

btw, welcome to the forums!

 

[Moonbear]

Yes, the pH noted for your TE buffer would mostly likely be for the final pH (that's the pH we use in our lab, but I of course can't say for certain we use the same protocol as your lab). You'll get very close to that if you adjust the pH of the Tris buffer, then add your EDTA. If the EDTA changes the pH appreciably, you may need to start with a different pH for your Tris to compensate for the addition of EDTA. You can correct the pH with HCl or NaOH, but purists would say you're then diluting your solution and weakening the ability of your buffer to do its job (I agree that if you need more than a few drops of either to correct the pH, then the problem is your starting buffer). Sometimes the best bet when in doubt is to call the person who you got the protocol from to clarify.

An aside on lab notebooks:
Absolutely, write everything in your lab notebook, legibly and in sufficient detail that the next person who needs to know what you've done can repeat it without having to guess. The other thing I sometimes have a hard time impressing upon my students is that the notes are supposed to be written WHILE you're doing the experiment, not when you're done for the day and going back trying to recall what you did. The lab notebook should be kept in a way that should you for any reason need to stop mid-way through a procedure (say you got sick and needed to leave and have someone else finish for you), anyone else in the lab should be able to look at your notebook and know exactly where you left off to pick it up for you. Think of it that your notebook isn't so much for your use as for that of the next person in the lab after you who has to use the protocols you've developed.

The other benefit of keeping good notes is just in the type of case you're asking about: you're not sure if you're following a recipe correctly (or even if you think you're certain about it, but still make a mistake), and/or your experiment doesn't work. If you have good notes of what you did, going through those notes with someone more experienced would enable them to help spot any errors in the protocol, or conversely, prove to them that you did everything correctly and the experiment really just isn't going to work that way. Especially when someone is new to a lab, when they do their first experiments and something doesn't work, it's really hard to know if the problem is with the new person's technique or with the experiment itself. Good notes will help your mentor sort through this with you.

 

[Monique]

I'm very detailed with keeping my lab-notebook. First I write out the whole experiment and while I'm doing it I mark every step, or every addition, and note down any deviations or observations.

At one point I was doing a LOT of PCRs and started noting that some 96 well plates were not performing very well, and actually got worse and worse. I was doing the exact same protocol as ever, the same DNA, the same solutions. When I put everything in perspective it turned out all PCRs with a product larger than 150-200bp were getting worse. The nature of the DNA caused it to degrade fast and left me with only short templates to PCR on.

It's important to have good book keeping in order to make a quick diagnosis.