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Rnase A for plasmid miniprep


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4 replies to this topic

#1 lotus

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Posted 23 July 2010 - 10:31 AM

Hello,
Our lab is using the qiagen miniprep kit. Somehow, the Buffer p1 is going to empty soon even though we still have a lot of columns and other buffers left. I can make the buffer P1 in the lab myself, but the question is what to do about the Rnase a to be added to the buffer. Qiagen charges $199 for a replacement vial of Rnase A.
Does anyone know of any cheaper alternatives out there?

#2 perneseblue

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Posted 23 July 2010 - 03:28 PM

Hello,
Our lab is using the qiagen miniprep kit. Somehow, the Buffer p1 is going to empty soon even though we still have a lot of columns and other buffers left. I can make the buffer P1 in the lab myself, but the question is what to do about the Rnase a to be added to the buffer. Qiagen charges $199 for a replacement vial of Rnase A.
Does anyone know of any cheaper alternatives out there?


If you want to save some money buy RNAse A powder (which isn't DNAse free). You can find RNAse from distributors like Fisher Scientific. Then follow the RNAse A preparation protocol which requires you to boil the RNAse solution.

1. dissolve RNase powder in 0.01M sodium acetate (adjust to pH 5.2)
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. Spin down at 1000rpm for 1min to remove denatured protein.
5. dispense in aliquots and store at -20C

Applichem sell RNAse A poweder
100mg for $47
500mg for $134
May your PCR products be long, your protocols short and your boss on holiday

#3 mole

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Posted 23 July 2010 - 11:00 PM

Hi,


We are purchasing just columns only and all solutions we are preparing our self.

So it is not suitable to add RNase A in P1 if you yourself preparing the solution, I usually mix it with Elution buffer at a concentration of (50 microgram/ml) and store that at -20 in aliquots.

Here after elution you should incubate your plasmid at 56 degree centigrade for 10 mins. (Don't worry nothing is going to happen your plasmid)

With Regards,

Mole .

#4 donny

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Posted 25 September 2010 - 11:28 PM

What is the RNase for actually? Is it really essential? The guys who last used the miniprep kit in my new lab left the P1 in the box instead of storing it in the fridge. They last used it a year ago! I wonder if the RNase is dead. I used it anyway and got my plasmid but I wonder if there will be any future problem.

#5 Kaioshin

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Posted 26 September 2010 - 04:55 PM

What is the RNase for actually? Is it really essential? The guys who last used the miniprep kit in my new lab left the P1 in the box instead of storing it in the fridge. They last used it a year ago! I wonder if the RNase is dead. I used it anyway and got my plasmid but I wonder if there will be any future problem.



Well...it degrades the RNA

Did you run your mini-prep'd plasmid on a gel? If so, and you didn't see any smearing - congratulations, the RNase seems to still be active (it's quite stable, as evidenced by the preparation method posted above).






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