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SAM target sgRNA cloning protocol – S. Konermann, Zhang lab, 2014
Optimized sgRNAs for any coding human gene can be found using our SAM Cas9
activator design tool:
http://sam.genome-engineering.org/database/
In order to clone the guide target sequence into the sgRNA(MS2) cloning backbone
(addgene #61424 ) or lenti sgRNA(MS2)_zeo backbone (addgene #61427), synthesize
two oligos of the following form. Both plasmids have the same overhangs:
Example oligo design: Note that the NGG PAM is not included in the designed oligos.
Oligonucleotide ordering tips: Standard de-salted oligos (usually the most inexpensive
synthesis) are sufficient for cloning. If not already resuspended, dilute each oligo to 100
μM in sterile water or TE.
Golden-Gate sgRNA cloning protocol
1. Oligo anneal
Component
Amount [ul]
Each oligo [100uM]
1
10X T4 ligase buffer (NEB)
1
T4 PNK (NEB)
0.5
H
2
0
6.5
Mix the components above and anneal in a thermal cycler with the following conditions:
37ºC for 30min
95ºC for 5 min
Ramp to 25ºC at 5ºC/min
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2. Golden Gate reaction
Add 90ul of PCR clean H
2
O to the finished oligo anneal from above to dilute it 1:10. Then
mix the following components:
Note: use BbsI enzyme for the non-lentiviral SAM sgRNA backbone (addgene #61424)
and BsmBI enzyme for the lenti SAM sgRNA (zeo) backbone (addgene #61427).
Component
Amount [ul]
2X rapid ligase buffer (Enzymatics)
12.5
BSA [20mg/ml] (NEB)
0.125
Restriction enzyme (BbsI for 61424 or BsmBI for 61427) (Fermentas FD)
1
T7 ligase (Enzymatics)
0.125
Diluted oligo anneal (1:10) from above
1
Backbone Vector [25ng/ul]
1
H
2
0
9.25
Run the following program on a thermal cycler:
37C for 5 min
20C for 5 min
! repeat for 15 cycles total
Transform 2ul of the golden gate reaction in Stbl3 (or other recombination deficient)
competent cells. Plate onto Ampicillin plates. In general, picking 2-3 colonies per guides
should be sufficient to ensure a correct clone.
Note: it is not necessary to perform a negative control golden-gate reaction (without
insert) as it will always contain colonies and is not a good indicator of cloning success.
