Hi Dawn! Thanks for watching and leaving a comment, we appreciate it! This was one of our older videos - we've since slowed down our videos so they are easier to follow. Thanks for your feedback!
No, you don't have to change media 24 hours after transfection. 5-8 hours after plating the transfection mixture, you should add FBS and Antibiotic to the transfected plates to allow the cells to recover. If the cells are in poor condition, your confluency may have been too low in your plates. We suggest having a confluency between 70-80%. Hope this helps!
WT adenovirus and recombinant adenovirus differ in replication competency. For laboratory safety, recombinant adenovirus is replication incompetent. Ready-to-use adenovirus provided by abm omit the early viral genes, E1 and E3, but still maintain the necessary viral packaging signals and inverted terminal repeat sequences required for viral transcription. A replication-incompetent (-E1/-E3) virus can therefore efficiently infect cells but, once the infection has taken place, it cannot produce new virions, unless amplified in packaging cell lines that express E1 gene products (such as HEK293 cells).
We use the packaging cell line that express E1a proteins, and grow the cells to ~70% confluency, and transfected the cells with the plasmids that contain Rep and Cap genes, adenoviral helper genes, and the gene of interest (i.e., 3 plasmid co-transfection). You can find the packaging protocol at: www.abmgood.com/pub/media/productdocument/document/AAV%20%20Packaging%20%20Protocol.pdf
Hi Alae, thanks for watching our video and leaving a comment! This was one of our earlier videos and we have since improved our video narration speed. If it helps, you can also go to the "Settings" (gear icon on the bottom right of the video) and "Playback Speed" to select a slower speed!
Great video. Would be great if you could slow down in speaking.
Hi Dawn! Thanks for watching and leaving a comment, we appreciate it! This was one of our older videos - we've since slowed down our videos so they are easier to follow. Thanks for your feedback!
Buddy, just decrease speed to .75
@@vinitparekh4843 I did that too
Crisp , Coherent , clear ❤
do I need to change the medium 24 hours after transfection？ I saw my cells is in poor conditon after transfection.
No, you don't have to change media 24 hours after transfection. 5-8 hours after plating the transfection mixture, you should add FBS and Antibiotic to the transfected plates to allow the cells to recover.
If the cells are in poor condition, your confluency may have been too low in your plates. We suggest having a confluency between 70-80%. Hope this helps!
what is the difference between adenovirus and WT adenovirus?
WT adenovirus and recombinant adenovirus differ in replication competency. For laboratory safety, recombinant adenovirus is replication incompetent. Ready-to-use adenovirus provided by abm omit the early viral genes, E1 and E3, but still maintain the necessary viral packaging signals and inverted terminal repeat sequences required for viral transcription. A replication-incompetent (-E1/-E3) virus can therefore efficiently infect cells but, once the infection has taken place, it cannot produce new virions, unless amplified in packaging cell lines that express E1 gene products (such as HEK293 cells).
4:50 transcap chelation
What are aav pseudotypes
pac man
Are you growing cells first with aav and then once its 70% confluent, you transfected it with the plasmid that contains the gene of interest?
We use the packaging cell line that express E1a proteins, and grow the cells to ~70% confluency, and transfected the cells with the plasmids that contain Rep and Cap genes, adenoviral helper genes, and the gene of interest (i.e., 3 plasmid co-transfection).
You can find the packaging protocol at: www.abmgood.com/pub/media/productdocument/document/AAV%20%20Packaging%20%20Protocol.pdf
video speed set a 0.75 i think. great vid tho
i set it too 1.25, too slow sometimes
freeze-thaw method is not the best option now we have better technology
interseting video but your speetch was realy fast
Hi Alae, thanks for watching our video and leaving a comment! This was one of our earlier videos and we have since improved our video narration speed. If it helps, you can also go to the "Settings" (gear icon on the bottom right of the video) and "Playback Speed" to select a slower speed!
You're spelling is realy bad
Good
Thanks for watching!
I wish the presenter had a better speech skills, somehow disturbed the video
slow down bro....