6 replies to this topic

[sagar]

hi guys

im facing difficulty in preparing rnase that works properly .i use the protocol in molecular cloning ,sambrook ,but my rnase doesnt work whatso ever time i try.is there any other method to prepare rnase solution from powder form.
how to dissolve it in water if its possible to-then how to adjust for pH ?
Can any one help plz?

[HomeBrew]

RNAse is a pretty hardy enzyme -- if it's not working, it's likely because of the way you're using it, not the way you're making it.  Under what conditions are you doing the digestion?  I usually dissolve RNAse in 50-100 mM Tris, pH 7.5, and do the digestions under similar conditions at 37°C for 30 minutes to one hour.

What are you digesting?  I suppose a protease could be getting in the way

[sagar]

HomeBrew, on Apr 2 2009, 08:46 AM, said:

RNAse is a pretty hardy enzyme -- if it's not working, it's likely because of the way you're using it, not the way you're making it.  Under what conditions are you doing the digestion?  I usually dissolve RNAse in 50-100 mM Tris, pH 7.5, and do the digestions under similar conditions at 37°C for 30 minutes to one hour.

What are you digesting?  I suppose a protease could be getting in the way

Im trying to perform a cloning reaction but for that i need pure form of plasmid manually without using a kit
and the rnase im preparing at a conc of 10mg/ml by dissolving rnase in 900microlt of 100mM sodium acetate(pH 5.2) and after heating for 15mins in waterbath at 100C ,i adjust the pH with 0.1 volume of tthe reaction ie 100microlt of tris -hcl(ph 7.4) .
i hope this is the correct method.but after the rnase treatment the rna contamination stays in the isolated plasmid.

[HomeBrew]

Ok, so I see how you make your 10 mg/ml stock.  But how do you do your digestions?  And, out of curiosity, why can you not use a plasmid isolation kit?

[sagar]

HomeBrew, on Apr 3 2009, 02:55 AM, said:

Ok, so I see how you make your 10 mg/ml stock.  But how do you do your digestions?  And, out of curiosity, why can you not use a plasmid isolation kit?

just add 5microlt of the rnase that should be sufficient to digest 100-800ng of rna completely.but i still see the rna contamination in gel even after increasing the rnase amount.
My supervisor does not want me to use kit but do manually perfectly
anyways thanks

[HomeBrew]

You need to give more detail.  What is your plasmid isolation protocol?  At what step and for how long do you do the RNAse digestion?  At what temperature is the digestion done?

[sagar]

HomeBrew, on Apr 4 2009, 07:50 AM, said:

You need to give more detail.  What is your plasmid isolation protocol?  At what step and for how long do you do the RNAse digestion?  At what temperature is the digestion done?

hi
i perform plasmid isolation using miniprep method and give rnase treatment for 1 hr at 37C
i tried to do digestion at 65C for 30mins also but  rna contaminationn stays and the amount observed in gel after rnase treatment is same as that without  rnase treatment