Cloning custom sgRNAs into Zhang lab plasmids
Updated 1/5/2016 04:32 AM
Introduction
Materials
Procedure
- Digest and dephosphorylate 5 μg of plasmid with BbsI for 30 min at 37°C.
Table1
A A | B B | |
1 1 | Amount | |
2 2 | Plasmid concentration (μg/μL) | 1 |
3 3 | Plasmid (μg) | 1 |
4 4 | Plasmid volume (μL) | 1 |
5 5 | FastDigest BbsI | 1 |
6 6 | FastAP | 1 |
7 7 | 10X FastDigest Buffer | 2 |
8 8 | ddH2O | 15 |
9 9 | Total (μL) | 20 |
- Gel purify digested plasmid using QIAquick Gel Extraction Kit and elute in EB. (See protocol.)
- Phosphorylate and anneal each pair of oligos:
Table2
A A | B B | |
1 1 | Number of pairs | 5 |
Table3
A A | B B | C C | |
1 1 | Amount per rxn (μL) | Master Mix (μL) | |
2 2 | 10X T4 Ligation Buffer | 1 | 5 |
3 3 | ddH2O | 6.5 | 32.5 |
4 4 | T4 PNK | 0.5 | 2.5 |
5 5 | Master Mix Total | 8 | 40 |
6 6 | Oligo 1 (100 μM) | 1 | |
7 7 | Oligo 2 (100 μM) | 1 | |
8 8 | Reaction Total | 10 |
Please use the T4 Ligation Buffer since the buffer supplied with the T4 PNK enzyme does not include ATP (or supplement to 1mM ATP).
Put the phosphorylation/annealing reaction in a thermocycler using the following parameters:
Table4
A A | B B | |
1 1 | 37°C | 30 min |
2 2 | 95°C | 5 min and then ramp down to 25°C at 5°C/min |
Dilute annealed oligos from Step 3 at a 1:200 dilution into sterile water or EB.
- Set up ligation reaction and incubate at room temperature for 10 min:
Table5
A A | B B | C C | |
1 1 | Amount | Master Mix (μL) | |
2 2 | BbsI digested plasmid concentration (ng/μL) | 50 | |
3 3 | BbsI digested plasmid (from step 2, ng) | 50 | |
4 4 | BbsI digested plasmid (volume) | 1 | 5 |
5 5 | 2X Quick Ligase Buffer | 5 | 25 |
6 6 | ddH2O | 3 | 15 |
7 7 | Master Mix Total (μL) | 9 | 0 |
8 8 | Oligo duplex (1:200 dilution) (from step 3) | 1 | |
9 9 | Quick Ligase | 1 | |
10 10 | Reaction Total (μL) | 11 |
- (optional) Treat ligation reaction with PlasmidSafe exonuclease to prevent unwanted recombination products.
Table6
A A | B B | C C | |
1 1 | Amount (μL) | Master Mix (μL) | |
2 2 | 10X PlasmidSafe Buffer | 1.5 | 7.5 |
3 3 | 10mM ATP | 1.5 | 7.5 |
4 4 | ddH2O | 1 | 5 |
5 5 | Master Mix Total | 4 | 20 |
6 6 | Ligation reaction (step 4) | 11 | |
7 7 | Reaction Total | 15 |
- Transformation
This protocol is for cloning PX330-based plasmids including PX458-462 - SpCas9 (or SpCas9n D10A nickase) + single guide RNA. It also applies to PX260 and PX334 - SpCas9 (or SpCas9n D10A nickase) + CRISPR array + tracrRNA.
It is copied here from information from the Zhang Lab made available on Addgene. Also see the CRISPR forum for any help needed.
To edit this protocol, sign in with Benchling, click the clock icon on the top right, and click the Clone From Version button.
Bolded values in spreadsheets below indicate ones that you should change. Mixture calculations will automatically update.