sgRNA small fragment assembly
Updated 6/26/2017 01:51 PM
Introduction
Materials
- Oligo pairs (100µM)
- 10 x T4 ligase buffer (Promega)
- T4 PNK (NEB)
- BsmBI (NEB)
- T7 DNA ligase (NEB)
Procedure
Oligo phosphorylation and annealing
- 1. Phosphorylate the 5' end of each oligo separately by treating with PNK - let following mixture sit for 1 hr at 37C:
- 1 μl oligo (100µM)
- 1 μl 10x ligase buffer
- 7 μl H2O
- 1μl T4 PNK
- Mix the 10 μl of each phosphorylated oligo together and bring the total volume to 200 μl with water
- Take 50 μl of the mixture and run it on the thermocycler using the "Anneal" program:
1. 96˚C for 6 min
2. 0.1˚C per second ramp down to 23˚C
3. Hold at 23˚C
Small fragment Golden Gate assembly
- Use 2 μl of the mixture to ligate into the sgRNA entry vector, pWS082:
- 2 μl of annealed oligos
- 0.5 μl of sgRNA entry vector
- 1 μl 10x ligase buffer
- 1 μl BsmBI
- 1 μl T7 DNA ligase
- Thermocylcer protocol
1. 42°C for 2min
2. 16°C for 5min
3. Repeat steps 1-2 (10x)
4. 60°C for 10min
5. 80°C for 10min
- Transform half the reaction mixture into E.coli
- Select non-GFP colony and inoculate in selective media
- Miniprep plasmid and send for sequencing
Comments
shouldn't the annealing oligos be as follows for cloning into pWS082 be as below?
5’ GACTTTNNNNNNNNNNNNNNNNNNNN 3’
3’ AANNNNNNNNNNNNNNNNNNNNCAAA 5’
5’ GACTTTNNNNNNNNNNNNNNNNNNNN 3’
3’ AANNNNNNNNNNNNNNNNNNNNCAAA 5’
can you please explain about the reverse primer (3`AAN...CAAA5`)? thanks
Oligos do not need to be phosphorylated. The gRNA entry vector contains phosphates on the ends during the Golden Gate reaction.
After selecting your guides in the ‘design CRISPR’ workspace, click the assemble button. This will prompt you to select an expression vector. If this is your first assembly, you will first need choose a plasmid from your Benchling folder. Select the plasmid pWS082 and choose 516 and 1541 as your start and end position, respectively, and click next. Title your assembly (e.g. gWS001), and add an additional ‘TT’ to the 5’ end of your guide sequence (reason for this described in suplementary figure S22 Lee et al., 2015, A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly) and click assemble. You can now copy the primer list into an excel sheet, or directly into IDT, and create a notebook entry for this assembly. Once the oligos arrive you will need to phosphorylate and anneal them.
This should look as follows:
5’ GACTTTnnnnnnnnnnnnnnnnnnnn 3’
3’ AAnnnnnnnnnnnnnnnnnnnnCAAA 5’