Paul.xu at 14:27, 6 October 2012

2012-10-06T14:27:18Z

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== Oligo Design ==

to be added 

== Cloning ==

=== Annealing Step ===

In PCR tube, mix the following: 1 ul 10X PCR buffer (no Triton X-100, no DMSO) 

2 ul sense (top) oligo (10 uM) 2 ul antisense (bottom) oligo (10uM) 

5 ul water to 10 final volume. 

Incubate the tube for 2 minutes at 95°C. 

Turn off PCR machine immediately and let the tube cool down to RT. 

Store the annealed oligos at 4 oC before ligation or store at -20 °C for long term storage. 

=== Vector Digestion ===

Digest vector with restriction enzymes, gel purify vector, elute with 15 ul water. 

Note: make sure you use fresh 0.5X TAE and wash electrophoresis tank before running a gel 

 

=== Ligation ===

Add 

5 ul annealed oligo mix to gel-purified vector. 

2 ul 10X ligation buffer or 15 ul 2X fast ligation buffer 

0.5 ul T4 DNA ligase, or T7 DNA ligase 

Incubate at RT for 1 ~ 2 hours or O/N 

 

=== Transformation ===

Add 50 ul STBL3 or Top10 competent cells to 10 ul ligation mix.

Incubate for 30 min on ice.

Heat shock at 42 oC for 1 min, cool on ice for 2 min.

Add 300 LB/Y or recovery medium and incubate at 37 oC (shaking, 750 rpm) for 30 min to 1 hour.

Spin down cells, plate on LB/Amp or LB/Kan plate. 

=== Screening ===

1. Make miniprep, digest with restriction enzymes. 

2. Sequencing.

[[Category:Protocols]]

ShRNA Cloning - Revision history

Paul.xu at 14:27, 6 October 2012