RNA extraction with the RNeasy Mini Kit - BUFFER RPE W/O ETH

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

Moderators: mchlbrmn, Abhijeet Bakre

RNA extraction with the RNeasy Mini Kit - BUFFER RPE W/O ETH

Postby bdunn10 » Jan 14 2016 7:23 pm

Hello,

1) I forgot to add 44ml of ethanol to the Buffer RPE (to make 55ml total) BEFORE adding 500µl of it to the RNeasy spin column. Will this effect the RNA extraction since I used 500µl Buffer RPE concentrate (not diluted with ethanol)?

2) If I added the 44ml of ethanol to the Buffer RPE after using 500µl of the Buffer RPE concentrate then is the bottle useless? Should I purchase a new Buffer RPE and add the ethanol first?

3) Was it okay to use 75% ethanol or should I have used 100% ethanol?

Thank you,
Brittany
bdunn10
newcomer
newcomer
 
Posts: 1
Joined: Jan 14 2016 7:14 pm

Re: RNA extraction with the RNeasy Mini Kit - BUFFER RPE W/O

Postby relaxin » Jan 15 2016 11:48 am

1. Without the EtOH, the RPE buffer will not work.

2. You have 10.5 ml RPE left after using 500 ul. You should add only 10.5/11 x 44 = 42 ml of Ethanol. If you added 44 ml Ethanol already, it may or may not work. You need to try with a sample that can be replaced. If it does not work, then order a new one or buy a new kit. You can also ask your neighbors and see if they have leftover RPE.

3. You should use 100% Ethanol. Some Ethanol contain other alcohol such as Propanol, they should be fine.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7174
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Postby r.rosati » Jan 16 2016 7:41 am

Regarding point 3 (having added 75% ethanol), this can reduce yields, as a washing buffer with too much water will very likely wash some RNA off the column. Maybe it'll still hold, but... you're definitely off the specs for the kit. As Relaxin suggested, you could try the kit on a sample you don't mind losing and see how it goes.
r.rosati
ModSquad
ModSquad
 
Posts: 2136
Joined: Nov 04 2002 10:23 am
Location: Brazil


Return to RNA Methods

Who is online

Users browsing this forum: Alexa [Bot] and 1 guest