TITLE: CRISPRon-GFP (based on pX459 plasmid) cloning of guides
AUTHORS: Ofer Barnea-Yizhar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was written in accordance in accordance with the Zheng Lab protocol and the following lessons learned from the following papers.</div></div>

[STEPS]
?. [Vector digest]
Digest  of an empty vector with (the enzyme is kept in -80 REVCO). Comprise the reaction as follows:  AB1Vector  (dervided from CRIPSRon) 1-1.5 ug ( X uL) 2 BbsI enzyme   1 uL 3 NEB buffer 2.1  5 uL 4 ddH2O  up to 50 uL 5 TOTAL  50 uL
1 µg
AB1Vector  (dervided from CRIPSRon) 1-1.5 ug ( X uL) 2 BbsI enzyme   1 uL 3 NEB buffer 2.1  5 uL 4 ddH2O  up to 50 uL 5 TOTAL  50 uL
?. [Vector digest]
Run for 1hr at, and another  at  for inactivation
37 °C
65 °C
?. [Phosphorylate and anneal each pair of oligosVector digest]
In a 0.2ml PCR tube prepare the annealing and phosphorylation reaction as follows:  AB1 FWD oligo (100uM) 1 uL2 REV oligo (100uM)   1 uL 3 10X T4 ligation buffer (NEB)   1 uL 4 T4 PNK (NEB)   0.5 uL 5 ddH2O  6.5 uL 6 TOTAL  10 uL
AB1 FWD oligo (100uM) 1 uL2 REV oligo (100uM)   1 uL 3 10X T4 ligation buffer (NEB)   1 uL 4 T4 PNK (NEB)   0.5 uL 5 ddH2O  6.5 uL 6 TOTAL  10 uL
?. [Phosphorylate and anneal each pair of oligosVector digest]
Run the reaction in a thermocycler according to the following parametersthen ramp down to 25oC at the rate of 5oC per min[see program OFER/CRISPRAN in the thermocycler ]
[20min]
[5min]
?. [Ligation reaction]
In a 0.2ml PCR tube prepare the ligation reaction as follows: [no need to check the concentration of the gel purified digested vector, just take a fix volume]   AB1 BbsI digested vector from previous step  5 uL 2Phosphorylated and annealed oligo duplex from previous step                   (1:250 dilution) 1 uL 3 10X T4 ligation buffer (NEB)   2 uL 4 ddH2O  11 uL 5 T4 Ligase (NEB)  1 uL
AB1 BbsI digested vector from previous step  5 uL 2Phosphorylated and annealed oligo duplex from previous step                   (1:250 dilution) 1 uL 3 10X T4 ligation buffer (NEB)   2 uL 4 ddH2O  11 uL 5 T4 Ligase (NEB)  1 uL
?. [Ligation reaction]
It is possible to prepare a stock of the T4 Ligase, buffer water and digested plasmid and aliquot 19ul to each 0.2ml PCR tube.
?. [Ligation reaction]
Put the reaction in a thermocycler set at  for 10-15min
25 °C
?. [Vector digest]
OPTIONAL - run a sample from the digested product on 1% agarose gel with an UNCUT control
?. [Transformation]
Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.
?. [Transformation]
Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture.
?. [Transformation]
Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
?. [Transformation]
Place the mixture on ice for 30 minutes. Do not mix.
For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.
?. [Transformation]
Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.
?. [Transformation]
Place on ice for 5 minutes. Do not mix.
?. [Transformation]
Pipette 950 µl of room temperature SOC into the mixture.
950 µl
?. [Transformation]
Place at 37°C for 60 minutes., shaking vigorously (250 rpm) or rotating.
Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.
?. [Transformation]
Warm selection plates to 37°C.  (Ampicilin)
Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.
?. [Transformation]
Mix the cells thoroughly by flicking the tube and inverting.
?. [Transformation]
Perform several 10-fold serial dilutions in SOC.
?. [Transformation]
Spread 50-100 µl of each dilution onto a selection plate
?. [Transformation]
Incubate overnight at 37°C
Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
?. [Sequence analysis]
Pick up to 4 colonies for each sgRNA clone (oligo pair) and touch a REPLICA plate and insert into 2ml liquid LB (in a 2phase 13ml tube) with the right selectable marker antibiotic (ampicillin or kanamycin)grow 2 colonies per gRNA, keep 4 on replica plates. Do not throw away the transformation plates until a positive clone is confirmed
?. [Sequence analysis]
Grow in arotating incubator for 6-18hrs (until the media opaque) grow 2 colonies per gRNA, keep 4 on replica plates. Do not throw away the transformation plates until a positive clone is confirmed
?. [Sequence analysis]
Use plasmid miniprep kit Send to sequencing using either the px459-U6-SEQ primer or the pLKO5.1 primer pLKO5.1 SEQ primer gactatcatatgcttaccgt  px459-U6-SEQ primer  caaggctgttagagagataa