Editing ShRNA Cloning

== Oligo Design<br>  ==

to be added<br> 

== Cloning<br>  ==

=== Annealing Step<br>  ===

In PCR tube, mix the following:<br>1&nbsp;ul 10X PCR buffer (no Triton X-100, no DMSO)<br> 

2 ul sense (top) oligo (10 uM)<br>2 ul antisense (bottom) oligo (10uM)<br> 

5 ul water to 10 final volume.<br> 

Incubate the tube for 2 minutes at 95°C.<br> 

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

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

=== Vector Digestion<br>  ===

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

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

<br>

=== Ligation<br>  ===

Add <br> 

5 ul annealed oligo mix to gel-purified vector.<br> 

2 ul 10X ligation buffer or 15 ul 2X fast ligation buffer<br> 

0.5 ul T4 DNA ligase, or T7 DNA ligase<br> 

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

<br>

=== Transformation<br>  ===

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.<br> 

=== <br>Screening  ===

1. Make miniprep, digest with restriction enzymes.<br> 

2. Sequencing. 

[[Category:Protocols]]