Download User Manual

Cellecta CRISPR Pooled sgRNA Libraries
www.cellecta.com
*Please see the PAC for primer sequences used for your particular library vector, or visit the Cellecta
website to find detailed information for standard vectors: http://www.cellecta.com/resources/vectorinformation/.
**IMPORTANT: During the PCR reaction, take a 5 µl aliquot from the tube after 10, 12, and 14
cycles and save it for the next step. The goal is to find the optimal cycle number in order to avoid
overcycling of PCR reactions, which can result in the generation of a longer fragment that
corresponds to a fusion double-sgRNA product.
2. The amplified sgRNAs are then analyzed on a 3.5% agarose-1XTAE gel (load 5 µl/lane). The
results should reveal a bright band of amplified sgRNA products. The goal of this analytical PCR
step is to optimize the starting amount of First Round PCR product and the number of cycles (if
necessary) in order to achieve equal intensities of a single band across all DNA samples from the
genetic screen.
3. Repeat second-round amplification of sgRNAs from each sample using the optimized volume of
First Round PCR product, 2 × 100 µl of Second Round PCR product per sample, and 12-18 cycles
of PCR. Set up 2 × 100 µl reactions for each sample containing an adjusted “equal” amount of
First Round PCR product (2 µl or more). Prepare a master mix according to the table below, for
each sample, where x is the volume of First Round PCR product:
x µl
First Round PCR Product
10 µl
Forward 2nd round PCR primer (10 µM)
10 µl
Reverse 2nd round PCR primer (10 µM)
4 µl
50X dNTP (10 mM each)
20 µl
10X Titanium Taq Buffer
152 - x µl
4 µl
200 µl
Deionized water
50X Titanium Taq
Total volume (Split into 2 x 100 µl reactions)
Perform PCR under the following cycling conditions.
94°C, 3 minutes
94°C, 30 seconds
65°C, 10 seconds
1 cycle
12-18 cycles (the number of cycles that worked
the best in the analytical PCR step)
72°C, 20 seconds
68°C, 2 minutes
4. Analyze the PCR products by gel-electrophoresis on a 3.5% agarose-1XTAE gel in order to ensure
equal yields of amplified sgRNAs for all samples. Combine amplified sgRNAs from the 2 × 100 µl
Second Round PCR reactions and purify the samples as follows:
a. Purify the PCR product with the QIAquick PCR purification kit (QIAGEN) following the
manufacturer’s protocol. In the last centrifugation step, use a centrifuge spin filter at
[email protected]
30 of 35
v1, 7/1/2015