Biology Forum › Molecular Biology › Prep. of 50X TAE buffer
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- May 2, 2009 at 9:29 am #11317
roniadamParticipantHi everyone:
I am a new person to mol. bio. lab and I want to prepare 50 X TAE buffer. I did the follwoing:
To make 1L of 50x TAE buffer:
242g Tris
100mL of 0.5M EDTA: This is prepared by (In 100 ml 1X PBS, Add 73.2 gram of EDTA)
57.1mL Glacial Acetic Acid
Add enough MQ H2O to dissolve solids to final volume of 1000mL.Is my work right??? The pH of PBS is about 7.6, Is this Ok for TAE buffer or I have to get the pH to 8??? If i should increase the pH, how can I do this step?
- May 2, 2009 at 3:48 pm #90524claudiupopaParticipant
Unfortunately im not familiar with making TAE but i can give you some general advice. In this case you have to take in consideration not only the pH of the PBS solution but also the effect of the acetic acid (which will be "pulling" your pH down into acidic) so if yout pH is to low try lowering the pH by adding a base such as NaOH
- May 3, 2009 at 12:20 am #90533canalonParticipant
EDTA is annoying to dissolve to say the least. It is slightly acidic, but disssolves only at pH 8. Also I usually do not prepare my solution in PBS but in water.
So take your PH meter, weigh your EDTA and add it to 80ml MQwater, then take a pipette, 10M NaOH and be patient. - May 3, 2009 at 6:06 am #90545roniadamParticipant
Thanks claudiupopa and Canalon
I am wondering why to add EDTA to 80 ml of MQwater rather than 100 ml? according to the protocol, I should add 100 ml of 0.5M EDTA. So how can I put it in 80 ml?
- May 4, 2009 at 1:47 am #90575canalonParticipant
No when you are preparing the 0,5M EDTA you first add 80ml of MQwater to the powder, and then try to dissolve the EDTA (it is a long and painful process, but it will eventually happen) by adding NaOH to the solution. And when all the powder has finally dissolved, you just complete to 100ml by adding MQwater again. Standard procedure to prepare a solution whose pH needs to be adjusted: leave space to add the solution that will adjust the pH.
- May 4, 2009 at 10:14 am #90581roniadamParticipant
Thanx Canalon
I rally appreciate your kind help. - May 7, 2009 at 4:30 pm #90676LifeTeinParticipant
My impression is that there are two kinds of EDTA, one is very acid, another is close to 7. Correct if I am wrong. You need to use the right one for your buffer.
- May 7, 2009 at 4:47 pm #90679MrMisteryParticipant
EDTA is a chemical. I guess it depends if you are discussing the protonated forms or the unprotonated forms. but for some reason, people don’t seem to distinguish. i can’t say really, i only use EDTA for DNA gel buffer, and that stuff is ready made.
- September 24, 2010 at 10:04 am #101458shekarParticipant
First you need to know preparation of 0.5m EDTA solution
Add 186.1 g EDTA (disodium, dihydrate, ) to 800 ml of ddH20. Add about 20g of NaOH pellets while stirring to bring the pH to 8.0. Add the last few grams slowly to avoid overshooting the pH. Note that the EDTA won’t completely dissolve until the pH is around 8,make the final volume to 1000ml with ddH2o. Filter with 0.5 micron filter and autoclave50 X TAE Buffer Preparation protocol (Tris-Acetate-EDTA)
242 gm – Tris base
57.1 mL – Acetic Acid
100mL – 0.5 M EDTA (shake vigorously before use)Add ddH2O to 1 Liter and adjust ph to 8.5 using KOH.
regards
shekar
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