Post Reply 
Thread Rating:
  • 0 Votes - 0 Average
  • 1
  • 2
  • 3
  • 4
  • 5
Sephacryl S400 columns to isolate monosomes
22-Aug-2013, 08:41 AM
Post: #1
Sephacryl S400 columns to isolate monosomes
Hi all I sent the following to Artseq technical support. If they answer I will post their response.

'In the ArtSEQ kit you suggest using S400 columns and I tried it but the protocol states that once opened the buffer should be allowed drip by gravity.....this definitely did not happen for me and I used 8 of the columns at the time. Only when I added the 1 x PLB did they start dripping. Also they all drip at different rates and so I am not sure that it good for some columns to be ready long for the others as they dry out quickly.

Also, can you really only load 100ul lysate per column? Not sure 100ul is enough (Artseg kit suggests 400-500ul) to do Riboseq with so does this mean several columns have to be used....very expensive.'
Find all posts by this user
Quote this message in a reply
13-Sep-2013, 10:01 AM
Post: #2
RE: Sephacryl S400 columns to isolate monosomes
Here is the reply from Artseq (I should point out that they did respond to me promptly but I have been too busy recently to post their reply).

Sure. The columns do not drip readily when you first open them. This can be due to air bubbles, compacted matrix, or other factors. It may take some encouragement by using the pad of a gloved finger to create pressure by covering the top of the column and pressing down lightly (may need to be repeated to get them started). (Also, insert usual disclaimer about having clean gloves to prevent contamination). I’ve also done the finger pressure method when some of the columns get stuck part way through the dripping process. The columns do drip more readily once the 1x PLB is added, but it still takes a while to pass the amount of 1x PLB stated in the protocol. The columns will also drip at different rates due to various different conditions mainly related how packed the matrix is. I would not encourage the columns to dry out before using them. Instead, just keep adding more 1x PLB buffer until the others are ready as well. You really just want to pass through enough 1x PLB to get rid of the buffer GE puts in their columns.

Isolating polysomes at the higher end of the kit's range (~50 million cells per lysate prep), one S400 column per lysate results in ~17ug of RNA. Assuming that in the lower range of cells (5 million), which is 1/10 the amount, you should expect ~ 1/10 the RNA, or ~1.7ug, which is still in the range of RiboZero. Most importantly, amount of material you recover is going to depend on cell line, type, organism, etc. Therefore, if you don't have enough RNA after one S400 column, you can easily do another S400 purification with leftover lysate and pool the two purifications without having to burn any of the ARTseq reagents.
Find all posts by this user
Quote this message in a reply
Post Reply 

Forum Jump:

User(s) browsing this thread: 1 Guest(s)

GWIPS-viz | Return to Top | Return to Content | Light (Archive) Mode | RSS Syndication