To clone pairs of sgRNAs into the pCRoatan dual-sgRNA expressing plasmid, synthetize two oligos of the following sequence:
sgRNA1-oligo |
AGCGGAAGACGCTCTAAAACNNNNNNNNNNNNNNNNNNNNCGGTGTTTCGTCCTTTCCAC |
sgRNA2-oligo |
GGCAGAAGACTAAAACNNNNNNNNNNNNNNNNNNNNCCGACTAAGAGCATCGAGACTGC |
In both oligos, the Ns are the reverse-complement of the 20bp target sequence. Standard desalted oligos can be used, resuspend the oligos to a final concentration of 100uM in water.
Step 1. Digest 4ug of pCRoatan plasmid with BsmbI for 2 hours at 37C:
4 ug |
pCRoatan plasmid |
5 ul |
10x Buffer Tango (Thermo Fischer) |
5 ul
|
10 mM DTT
|
2 ul
|
BsmBI (Thermo Fischer)
|
X ul
|
H2O
|
50 ul
|
Total volume
|
Step 2. Gel purify the digested plasmid using a QIAquick Gel Extraction Kit. A ~1kb filler should visible on the gel if the digestion was successful. Extract the top, ~7.5kb band.
Step 3. Amplify the dual-promoters using the two oligos as primers:
1 ul
|
100 uM sgRNA1-oligo
|
1 ul
|
100 uM sgRNA2-oligo
|
1 ul
|
10 ng pDU6 template
|
5 ul
|
10x KOD buffer (Millipore)
|
5 ul
|
DMSO
|
4 ul
|
MgSO4
|
2 ul
|
KOD
|
X ul
|
H2O
|
50 ul
|
Total volume
|
Put the PCR reaction in a thermocycler and run the following program:
- 95C for 5 mins
- 95C for 30 s
- 55C for 30 s
- 72C for 30 s
- Cycle to step 2 for 20 cycles
- 72C for 30 s
Step 4. Purify the PCR reaction using the QIAquick PCR purification kit and digest using BbsI for 2 hours at 37C:
4 ug
|
PCR product
|
5 ul
|
10x NEBuffer 2.1 (NEB)
|
2 ul
|
BbsI (Thermo Fischer)
|
X ul
|
H2O
|
50 ul
|
Total volume
|
Step 5. Gel purify the ~1.2kb PCR product containing the sgRNAs and the two U6 promoters using a QIAquick Gel Extraction Kit.
Step 6. Set up ligation and incubate at room temperature for 1 hour:
X ul
|
100 ng of digested pCRoatan
|
X ul
|
40 ng of digested PCR product
|
2 ul
|
10x T4 DNA ligase buffer (NEB)
|
1 ul
|
T4 DNA ligase (NEB M0202T)
|
X ul
|
H2O
|
20 ul
|
Total volume
|
Step 7. Transform into competent cells. Recombination-deficient bacteria must be used to prevent plasmid recombination. We use Endura electrocompetent cells (Lucigen #60242-2) for this transformation. Plate the transformation on Amp-Zeo-LB low salt agar plates.