Editing PiggyBacHR Genome Eng (section)

== Preparation of Homology Arms ==

<br> 5’ Homology Arm (5’HA)<br>1. PCR the 5’ HA and add any restriction site you choose from the 5’ MCS except BsaI to the forward primer. Add a BsaI site and the sequence GTTAA to the 3’ end of the reverse HR primer. Homology arms should be between 1 and 0.5 Kb. The 5’ HA cannot contain a BsaI site. 

5’ HA forward primer sequence 

Restriction Site in 5’MCS of PBHR vector 

5’gccgcc'''XXXXXX'''--------------------3’ 

extra Sequence&nbsp;&nbsp;&nbsp; sequence of your 5’HA (~20 bp) 

<br>5’ HA reverse primer sequence 

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; '''BsaI site''' &nbsp; &nbsp;&nbsp; <u>PiggyBac TTAA</u>

5’ gaagaa'''GGTCTC'''g<u>TTAA</u>-------------3’ 

extra Sequence&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; sequence of your 5’HA (~20 bp) 

2. Run out the PCR product on an agarose gel.<br>3. Cut of the band of the correct size and isolate the fragment.<br>4. Cut the fragment with the 5’MCS enzyme you chose and with BsaI.<br>5. Clean up the reaction and quantify the DNA. 

3’ Homology Arm (3’HA) 

1. PCR the 3’HA and add the BspQI site to the forward primer. Add any site you wish from the 3’ MCS except BspQI to the reverse primer. Homology arms should be between 1 and 0.5 Kb. The 5’ HA cannot contain a BspQI site. 

3’HR forward primer sequence 

BspQI Site Sequence of your 3’HA (~20 bp) 

5’taataa'''GCTCTTC'''<u>TTAA</u>------------------------------3’ 

extra sequence&nbsp;&nbsp;&nbsp; <u>PiggyBac TTAA</u> 

<br>3’HR Reverse primer sequence 

Restriction site from the 3’ MCS 

ttcttc'''XXXXXX'''------------------------------------ 

extra sequence&nbsp;&nbsp;&nbsp; sequence of your 3’HA(~20bp) 

2. Run out the PCR product on an agarose gel.<br>3. Cut of the band of the correct size and isolate the fragment.<br>4. Cut the fragment with the 3’ MCS enzyme you chose and with BspQI.<br>5. Clean up the reaction and quantify the DNA.