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Aug 07, 2018

Version 2

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# Cloning guides to lentiCRISPR v2 V.2

DOI

https://dx.doi.org/10.17504/protocols.io.sfvebn6

Amit Weiner<fnref n="1" />

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Amit Weiner

University of Toronto

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DOI: https://dx.doi.org/10.17504/protocols.io.sfvebn6

**Protocol Citation:** Amit Weiner 2018. Cloning guides to lentiCRISPR v2. protocols.io
https://dx.doi.org/10.17504/protocols.io.sfvebn6

**License:** This is an open access protocol distributed under the terms of the **Creative Commons Attribution License**, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

**Protocol status:** Working

We use this protocol and it's working

Created: August 07, 2018

<footnote n="1">¹University of Toronto</footnote>
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**Last Modified:** August 30, 2018

**Protocol Integer ID:** 14549

**Keywords:** guides to lenticrispr v2, lenticrispr v2, cloning guide

Troubleshooting
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## Vector preparation:

1. Digest the lentiCRISPRv2 vector:

3µg vector

2µl 10X Tango buffer (Thermo Fisher)

1µl 20mM DTT

1-1.5µl Esp3I (Thermo Fisher)

Water to 20µl

In a thermocycler:

37°C for 4 hours, inactivate at 65°C for 20 mins, keep at 4°C.

**SKIP** the alkaline phosphatase step.

2. Use a gel purification/PCR cleanup kit (Qiagen) **WITHOUT** running on a gel.

Optional: run ~200ng of the purified vector on a gel to verify digestion.

## Guide insert preparation:

3. Anneal and phosphorylate gRNA oligos pair:

1µl of each oligo (100µM stock)

1µl 10X T4 ligation buffer (not PNK buffer)

0.5µl T4 PNK

Water to 10µl

In a thermocycler: 37°C for 30 mins, 95°C for 5 mins, ramp down to 25°C at 0.1°C/sec (or 5-6°C/min).

Optional: keep at 4°C.

4. Serially dilute the annealed oligos to 1:500

## Ligation:

5. Out of ligation at a vector:insert molar ratios of 1:5, 1:10, 1:20, I found that 1:5 works best.

50ng vector

1.5µl 10X T4 ligation buffer (NEB)

1µl T4 ligase (NEB)

2µl diluted oligos (1:500)

Water to 15µl

Incubate at RT for 1-2 hours.
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Transformation:

6 Transform 5µl of the ligation reaction to 50µl Stbl3 chemically competent cells.

If you incubate the Stbl3 cells at 30°C the colonies will be VERY small so look for them carefully. Incubating them at 37°C didn't result in LTR recombination in my hands.