Tweet
Hi,
I am attempting to put together a set of adapters and blocking oligos for a multiplexed exome-capture experiment. I am following this: http://openwetware.org/images/1/10/Geller_exome.pdf
And a couple protocols from Roche/Bioos/other .
My questions are: 1) What modifications are necessary for the oligos that make the adapters? I have seen both with and without a penultimate phosphorothioate bond on one strand. Is the phosphorothioate bond necessary? What is it's purpose.
(There is also a 5' phos modification, I know this is necessary.)
2) For the blocking oligos, is there a preference in the 3' extension block? inverted-dT, dideoxy-C, or C3 spacer? I would like to use the C3 spacer since it is less expensive.
3) For both the blocking oligos & the adapters, what level/type of purification is required? HPLC or PAGE? I am assuming HPSF is in sufficient? How about "QuickLC"?
Thank you for your input!
I am attempting to put together a set of adapters and blocking oligos for a multiplexed exome-capture experiment. I am following this: http://openwetware.org/images/1/10/Geller_exome.pdf
And a couple protocols from Roche/Bioos/other .
My questions are: 1) What modifications are necessary for the oligos that make the adapters? I have seen both with and without a penultimate phosphorothioate bond on one strand. Is the phosphorothioate bond necessary? What is it's purpose.
(There is also a 5' phos modification, I know this is necessary.)
2) For the blocking oligos, is there a preference in the 3' extension block? inverted-dT, dideoxy-C, or C3 spacer? I would like to use the C3 spacer since it is less expensive.
3) For both the blocking oligos & the adapters, what level/type of purification is required? HPLC or PAGE? I am assuming HPSF is in sufficient? How about "QuickLC"?
Thank you for your input!