9 replies to this topic

[sagar]

hi
I have been trying to prepare RNase (10mg/ml)by dissolving it i sodium acetate (5.2)and adjusting the ph with addition of tris 7.4 ,but every time i prepare like this i end up with no result. After running the gel i still get rna contamination.Is there any other method of preparing Rnase?

plz any1 can help?
Thanks in advance

Edited by sagar, 26 February 2009 - 06:07 AM.

[gebirgsziege]

Why are you using sodium acetate? I have not done this for some time, but I think I used pure water to dissolve.....

[Wolverena]

sagar, on Feb 26 2009, 09:05 AM, said:

hi
I have been trying to prepare RNase (10mg/ml)by dissolving it i sodium acetate (5.2)and adjusting the ph with addition of tris 7.4 ,but every time i prepare like this i end up with no result. After running the gel i still get rna contamination.Is there any other method of preparing Rnase?

plz any1 can help?
Thanks in advance

What concentration was the sodium acetate? Was it 0.01M?

[sagar]

gebirgsziege, on Feb 26 2009, 08:25 PM, said:

Why are you using sodium acetate? I have not done this for some time, but I think I used pure water to dissolve.....

hi
how to prepare using water and how to adjust the pH ? I think rnase will precipitate in neutral ph?
can u give me the protocol?

[sagar]

Wolverena, on Feb 26 2009, 09:25 PM, said:

sagar, on Feb 26 2009, 09:05 AM, said:

hi
I have been trying to prepare RNase (10mg/ml)by dissolving it i sodium acetate (5.2)and adjusting the ph with addition of tris 7.4 ,but every time i prepare like this i end up with no result. After running the gel i still get rna contamination.Is there any other method of preparing Rnase?

plz any1 can help?
Thanks in advance

What concentration was the sodium acetate? Was it 0.01M?

hi there

yes i have been using 0.01M sodium acetate
how can i modify my protocol.
Can u plz tell me the optimum pH for rnase?

[Wolverena]

I used the protocol for preparing RNase A from Sambrook, which seems to work fine. An important step is to boil the RNase before you use it. The protocol is as follow:
1. dissolve RNase in 0.01M sodium acetate
2. heat to 100C for 15min, cool down at room temperature
3. adjust pH by adding 0.1vol of 1M Tris-Cl (ph 7.4)
4. dispense in aliquots and store at -20C

The optimum working range for RNase A is between pH 7 and 8 (I think).

If that protocol doesn't work, it might be worth to buy pre-made RNase A from Invitrogen.

[sagar]

Wolverena, on Feb 26 2009, 11:54 PM, said:

I used the protocol for preparing RNase A from Sambrook, which seems to work fine. An important step is to boil the RNase before you use it. The protocol is as follow:
1. dissolve RNase in 0.01M sodium acetate
2. heat to 100C for 15min, cool down at room temperature
3. adjust pH by adding 0.1vol of 1M Tris-Cl (ph 7.4)
4. dispense in aliquots and store at -20C

The optimum working range for RNase A is between pH 7 and 8 (I think).

If that protocol doesn't work, it might be worth to buy pre-made RNase A from Invitrogen.

thanks i have been using the same protocol but anyways its not working maybe somehing is wrong with the enzyme powder.

[Wolverena]

sagar, on Feb 27 2009, 11:52 AM, said:

thanks i have been using the same protocol but anyways its not working maybe somehing is wrong with the enzyme powder.

that could be. Sorry I couldn't help you.

Good luck....

[gebirgsziege]

I just added the water....like it was suggested at the Data sheet.....when Sigma uses water to test it, I thought it will be fine for me
It works fine in my applications (use it in genomic DNA extraction) and the pH of my working solutions is in the range where the activity should be fine, so I never thought of dissolving it anywhere else   maybe I should the next time?????

[phage434]

The powder is lyophilized from solution, which will contain buffers.  Adding water reconstitutes the buffers as well as the protein.