Find content
Male
Yes, prot K degrades RNAses. It's better to add RNAse first and then prot K.
Read this: http://www.ncbi.nlm....v/pubmed/153663
You are not the first person to ask this question.
http://www.protocol-...osts/21549.html
But also keep in mind that digestion of DNA by DNAse (in contaminated samples, tips, microtube) is not as rapid as digestion of RNA with RNAses. Therefore, you don't have to worry about that and you have time to add your prot k and RNAse. Also, prot k might need time to degrade RNAse, so even if you add them together, RNAse A will digest your RNA before prot k digests it. You need to realize you are adding non-cellular RNAse A and prot k. They are excess in your sample.
You can also try this by yourself. Extract total RNA and prepare these samples:
1- total RNA
2 - total RNA + RNAse A, incubate (10-30 min) + prot K
3 - total RNA + prot k + RNAse
run on gel and check if you see any RNA smear or 18s/28s bands in the 3rd sample. If you do then prot k degrades your RNAse, although I doubt it will completely do that.
#144910 Does Proteinase K digest RNAse in lysis buffer?
Posted
on 09 November 2012 - 04:24 AM
#139418 Preparation of bacterial cells for SEM and TEM
Posted
on 14 August 2012 - 07:58 AM
Thank you for your help!! 
#139092 observing mouse tongue
Posted
on 08 August 2012 - 01:56 AM
"pinned its limbs on sponge board "
What!
No one in there right mind is going to give you ethics approval for such a cruel procedure! That poor mouse will have numerous injuries from being pinned out each week. What kind of person even suggests sticking pins in a live mouse!!! Can you imagine waking up from anaesthetic with big ol' pin holes in your arms?
Okay, personal outrage aside, here is what I would do:
- using an inhaled anaesthetic (e.g. methoxyfluorane), wait until the mouse appears drowsy and has stopped wondering around the jar, but isn't fully under
- take her out, and gently (using blunt forceps) pull her tongue out and have a look, you'll have to be really careful to hold her tongue lightly so you don't hurt or bruise it
If you are holding her correctly, you should have no trouble with this. If you are using a particularly feisty mouse strain, you may need to knock the mouse completely out, but even so if you go easy on that anaesthetic they will recover well.
I use this method to take saliva samples from mice, every two days. They appear to tolerate the anaesthetic well (in fact I wonder if some take it too well as they seem less and less fussed about getting in the jar each time, little addicts!).
What!
No one in there right mind is going to give you ethics approval for such a cruel procedure! That poor mouse will have numerous injuries from being pinned out each week. What kind of person even suggests sticking pins in a live mouse!!! Can you imagine waking up from anaesthetic with big ol' pin holes in your arms?
Okay, personal outrage aside, here is what I would do:
- using an inhaled anaesthetic (e.g. methoxyfluorane), wait until the mouse appears drowsy and has stopped wondering around the jar, but isn't fully under
- take her out, and gently (using blunt forceps) pull her tongue out and have a look, you'll have to be really careful to hold her tongue lightly so you don't hurt or bruise it
If you are holding her correctly, you should have no trouble with this. If you are using a particularly feisty mouse strain, you may need to knock the mouse completely out, but even so if you go easy on that anaesthetic they will recover well.
I use this method to take saliva samples from mice, every two days. They appear to tolerate the anaesthetic well (in fact I wonder if some take it too well as they seem less and less fussed about getting in the jar each time, little addicts!).
