Which overhangs your backbone needs. The oligos below, the order-form text and the printed sheet all follow this choice, so set it before you order.
Type or paste the protospacer, 20 nt, 5′ to 3′, without the NGG PAM. Everything else follows as you type, and the sheet stays in this browser for next time.
| Target gene | # | Protospacer | Cloning oligos | Status |
|---|
One guide per line. Separate fields with a tab, comma or semicolon: the longest field that reads as DNA is taken as the sequence, the first other field as the gene. A line with no sequence in it is reported, not guessed at. Rows already here are kept.
Name and sequence, tab separated, in the order shown above.
CACCG / AAAC. BsmBI (Esp3I) leaves the same overhangs on lentiCRISPRv2,
lentiGuide-Puro and lentiCRISPRv1, so one oligo pair serves all three. Zhang lab protocol
rev20140722. pX330 takes the same oligo pair but is cut with BbsI (BpiI), not
BsmBI — check which enzyme your backbone needs before digesting. This reproduces that
protocol exactly, including the caccg prefix on guides that already begin
with G. If your vector convention drops that extra g, edit the sequence by hand before
ordering.
TTG / GTTTAAGAGC. From a shared annealing-oligo ordering spreadsheet
(annealing_oligo_ordering.xlsx, sheet Sheet1). The set is named
after its overhangs, not after a backbone, so confirm which vector and which enzymes you
need before digesting. Column D of that sheet is the top oligo,
=CONCATENATE("ttg",B,"gtttaagagc"). Column E is headed reverse complement
of bottom oligo and reads =CONCATENATE("cttgttg",B,"gtttaagagctaa"), so
the oligo you actually order is its reverse complement, which is what the worked example
pair in that sheet spells out.
The NGG PAM is never part of the oligos, in either set. Standard de-salted synthesis is enough; resuspend to 100 µM in sterile water or TE.
Nothing here leaves your computer. The sheet is stored in this browser only.