Time after transfection and puromycin selection using PX459

[Jerry Xu]

I'm curious about time issues about transfection and puromycin selection using PX459

1. After transfection of the sgRNA plasmids and ssODN, how long should I wait? 

   Feng Zhang protocol suggests 24 hrs before checking transfection efficiency.

   How long it takes for sgRNA/Cas9 start to work to edit genome?

2. To add puromycin for selection, I directly add additional 500ul medium containing puromycin to the original medium? Or should I dissociate the cells by trypsin and change for a new plate? 

3. How long should I wait after adding puromycin?  Feng Zhang protocol suggests 48 hrs-72 hrs.

4. I'm trying to test first in HEK293FT and then neuronal line SH-SY5Y. Maybe waiting time mentioned above could be different for different cell lines?

Hope to hear some experience.

Thx!

[Rocamar]

Hi jerry

Le 2 oct. 2015 à 04:51, Jerry Xu <gerryb...@gmail.com> a écrit :

I'm curious about time issues about transfection and puromycin selection using PX459

1. After transfection of the sgRNA plasmids and ssODN, how long should I wait? 

   Feng Zhang protocol suggests 24 hrs before checking transfection efficiency.

   How long it takes for sgRNA/Cas9 start to work to edit genome?

When I insert a reporter gene (RFP) at the cut site ...I see the red cells as soon as 12 hours..don t know if this is the transient expression of the episomal vector. 

2. To add puromycin for selection, I directly add additional 500ul medium containing puromycin to the original medium? Or should I dissociate the cells by trypsin and change for a new plate? 

I transfect in 6 wells...24h later, I trypsin the cells u2os and replate in fresh medium containing puromycin in 10cm plate (maybe a 140 cm is better to growth single clones). I wait until the ctl cells have died (2days usually) . Then I remove the puromycine (to avoir the intégration of the px459. Then wait for single colony to growth

This work pretty well..basically it takes around 40 day from the initial transfectiom to growth, analyse and store KO KI clones.

3. How long should I wait after adding puromycin?  Feng Zhang protocol suggests 48 hrs-72 hrs.

4. I'm trying to test first in HEK293FT and then neuronal line SH-SY5Y. Maybe waiting time mentioned above could be different for different cell lines?

For different cell lines optimize the puromycine concentration doing a killing curve .

Hope to hear some experience.

The technique is amazingly ..incroyable:-)

Didier

Thx!

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.
For more options, visit https://groups.google.com/d/optout.

[Jerry Xu]

Thanks a lot! Very detailed

[Joe Miano]

We transfect and select with optimal dose of puro immediately for 3-4 days and then do bulk PCR on same plate to see whether we get editing before proceeding.