Ampure XP buffer

[cli]

Hi, all,

I just want to know if 10% PEG 8000 and 1.25 M NaCl in final concentration would work for Ampure XP or what is the optimal concentration. In the manual, it said that only fragment > 100bp are retained. If I want to keep fragments ~ 50 bp, could I make it work by just increasing the salt/PEG concentration? Another question: can the beads still be used after 10 min cooking at 95 C? I know the best way is to test those by myself, but I'd like to hear from anyone already had some experience. Thanks in advance.

[Heisman]

I'm not sure about heating the beads a lot, but if you have say 12% PEG at 1.25 M NaCl you might be able to keep stuff at 50bp. Certainly I think at 15% PEG you can keep it. 10 will be a bit too low from personal experience. I don't think it's necessary to increase the salt concentration further.

[cli]

Thank you, Heisman. I will test 15% PEG and I will post the results.

[ECO]

In my experience, you'll have to test with your specific lot/manufacturer of PEG. Molecular weight and the distribution of sizes differ a fair amount, and can affect the cutoff.

There are several papers around that describe knock off buffers (and I think they are all in this thread: http://seqanswers.com/forums/showthread.php?t=3752 )...but if you're looking for a specific cutoff, the likelihood that you'll have to test your own buffer is extremely high.

[PigletIA]

Thank you, Heisman. I will test 15% PEG and I will post the results.

Hi, Cli, I'm also interested in keeping fragments down to 60nt. Mine is fragmented RNA 60~200nt. I heard the RNAClean XP is the same beads with AMPure XP, just different buffer. So I'm curious if the increasing concentrations of PEG worked out for you. Thanks!

[cli]

Hi, PigletIA, I have tested 15% PEG for SPRI cleanup. The 100 bp fragment was kept but not the 50 bp fragment. I didn't try higher concentration of PEG.

[PigletIA]

Hi, PigletIA, I have tested 15% PEG for SPRI cleanup. The 100 bp fragment was kept but not the 50 bp fragment. I didn't try higher concentration of PEG.

This is good to know, Cli! I guess it's not gonna be easy otherwise they've already figured it out...
Thanks a lot!
Lan

[illuminauser]

SpriSelect

Does Anyone have suggestions for Ratios to use for SpriSelect if I want to target 520bp range for cDNA?

[xiaozhe]

XP and Axygen buffer Maldi analysis

Hi, i had used Maldi mass to detect two types of buffer. the one is XP buffer and
the other is axygen buffer. Here is the result:





everyone could tell me the results whether contained PEG8000 and PEG2000? or had other answer?