problems in lentiviral transduction - time to collect, ratio for transfection (Apr/13/2011 )
hi guys, i am new to this experiment so i met lots of problems and want you to help me.
1. i insert my target gene, a 2.5kb fragment into pCSII-EF-IRES-hrGFP, which is about 10.4 kb. finally i got positive clones and confirmed them by enzyme digest. however, when i tried to get midi-preps i failed, nearly no DNA extracted. someone suggested me re-transform ecoli and pick the colony and directly do midiculture. i dont know why.
2. at what time point to collect supernatant containing viral particles? 36h? 48h? 60h? 72h? once or twice? if i save the supernatant at 4c for one night, is it ok for the activity of virus? what is the peak for virus production? is it true that virus will be inactivated at 37c for 24hrs?
3. I used pCMV-delta8/9 as packaging plasmid and pVSV-G as envelope plasmid, and the ratio is 10:9:1 (pCSII:packaging:envelope). I dont know if it is correct. I saw other ratios and dont understand what is the difference.
a lot of questions, sorry. thanks in advance.
gyma on Wed Apr 13 07:08:43 2011 said:
hi guys, i am new to this experiment so i met lots of problems and want you to help me.
1. i insert my target gene, a 2.5kb fragment into pCSII-EF-IRES-hrGFP, which is about 10.4 kb. finally i got positive clones and confirmed them by enzyme digest. however, when i tried to get midi-preps i failed, nearly no DNA extracted. someone suggested me re-transform ecoli and pick the colony and directly do midiculture. i dont know why.
2. at what time point to collect supernatant containing viral particles? 36h? 48h? 60h? 72h? once or twice? if i save the supernatant at 4c for one night, is it ok for the activity of virus? what is the peak for virus production? is it true that virus will be inactivated at 37c for 24hrs?
3. I used pCMV-delta8/9 as packaging plasmid and pVSV-G as envelope plasmid, and the ratio is 10:9:1 (pCSII:packaging:envelope). I dont know if it is correct. I saw other ratios and dont understand what is the difference.