cDNA cloning & expression - use ORF or full-length cDNA? - (Jan/29/2009 )
Hi All,
Please can someone help 
I want to express a protein in HEK293 cells via transfection in order to do functional studies (not for purification etc) so I dont think I want it tagged etc etc. But do you use the full length cDNA or the ORF clone? Both are available. I have no clue - please help!!
I am just interested in maximal expression of the active protein and will assay the cells directly for function.
Thanks in advance!
We used to express the ORF only. Including a Kozak sequence in front of the start codon may helps.
As suggested, use ORF.
Remember transient transfection will give you something like 1000x overexpression of the protein.
Hi,
sorry to bump in with not-exactly-related to the original question. But i have a question about transfection of retroviral vector using fugene6. found an exact post with the exact prob but the link was broken so i couldn't follow on that. Here's the situation:
Need to transfect an LZRS-ires-egfp (~13kb) plasmid into 293GP cell;
Did:
1. optimizing transfection using (ugDNA:ulFugene) 2:9, 3:9, 4:9, 5:9
2. optimize again using (ugDNA:ulFugene) 2:3, 2:6, 2:9, 2:12
all of these in 6-well plate with 40-50% cell confluency. The transfection results was very2 low as seen by microscopy while FACS showed it was ~20-30%. A control transfectin using pEGFPc1 (1ug:9ul) have better results (~80% by microscopy, though funny that FACS says it was just ~40%).
prep of dna:fugene mix and transfection were as follows:
- optimem + fugene (to final vol. 100ul) - incubate RT 5 min,
- optimem/fugene mix + DNA - incubate RT 1 hr
- remove overnight medium from plate and dropwise mix onto cell
- incubate at RT 1 hr with tilting of plate every 15 minutes
- top wells with 2ml complete DMEM medium (10% fcs + antibiotic) and incubate 48hrs before microscopy or facs-ing
Have any idea how else should i troubleshoot the transfection? Really appreciate for any feedbacks.
Thanks in advance.
shasa753 on Mar 1 2009, 07:51 PM said:
sorry to bump in with not-exactly-related to the original question. But i have a question about transfection of retroviral vector using fugene6. found an exact post with the exact prob but the link was broken so i couldn't follow on that. Here's the situation:
Need to transfect an LZRS-ires-egfp (~13kb) plasmid into 293GP cell;
Did:
1. optimizing transfection using (ugDNA:ulFugene) 2:9, 3:9, 4:9, 5:9
2. optimize again using (ugDNA:ulFugene) 2:3, 2:6, 2:9, 2:12
all of these in 6-well plate with 40-50% cell confluency. The transfection results was very2 low as seen by microscopy while FACS showed it was ~20-30%. A control transfectin using pEGFPc1 (1ug:9ul) have better results (~80% by microscopy, though funny that FACS says it was just ~40%).
prep of dna:fugene mix and transfection were as follows:
- optimem + fugene (to final vol. 100ul) - incubate RT 5 min,
- optimem/fugene mix + DNA - incubate RT 1 hr
- remove overnight medium from plate and dropwise mix onto cell
- incubate at RT 1 hr with tilting of plate every 15 minutes
- top wells with 2ml complete DMEM medium (10% fcs + antibiotic) and incubate 48hrs before microscopy or facs-ing
Have any idea how else should i troubleshoot the transfection? Really appreciate for any feedbacks.
Thanks in advance.
Sounds like your DNA is not very clean. Fugene and 293 cells should be quite easy...normally, I use 2ul of fugene per microgram of DNA...and I use PBS. I try to stay away from optimem as it does contain some protein (~15ug/ml). You should see a fine hazy consistency after you add the fugene to the DNA (appears in a couple minutes or so, especially if you are transfecting ug of DNA) ...and do not remove the media before you add the fugene. Just add it to the media, swirl a few times and that's it. You actually need the media to disperse the DNA around or you will not get an even transfection.
good luck
NemomeN on Mar 2 2009, 04:17 PM said:
shasa753 on Mar 1 2009, 07:51 PM said:
sorry to bump in with not-exactly-related to the original question. But i have a question about transfection of retroviral vector using fugene6. found an exact post with the exact prob but the link was broken so i couldn't follow on that. Here's the situation:
Need to transfect an LZRS-ires-egfp (~13kb) plasmid into 293GP cell;
Did:
1. optimizing transfection using (ugDNA:ulFugene) 2:9, 3:9, 4:9, 5:9
2. optimize again using (ugDNA:ulFugene) 2:3, 2:6, 2:9, 2:12
all of these in 6-well plate with 40-50% cell confluency. The transfection results was very2 low as seen by microscopy while FACS showed it was ~20-30%. A control transfectin using pEGFPc1 (1ug:9ul) have better results (~80% by microscopy, though funny that FACS says it was just ~40%).
prep of dna:fugene mix and transfection were as follows:
- optimem + fugene (to final vol. 100ul) - incubate RT 5 min,
- optimem/fugene mix + DNA - incubate RT 1 hr
- remove overnight medium from plate and dropwise mix onto cell
- incubate at RT 1 hr with tilting of plate every 15 minutes
- top wells with 2ml complete DMEM medium (10% fcs + antibiotic) and incubate 48hrs before microscopy or facs-ing
Have any idea how else should i troubleshoot the transfection? Really appreciate for any feedbacks.
Thanks in advance.
Sounds like your DNA is not very clean. Fugene and 293 cells should be quite easy...normally, I use 2ul of fugene per microgram of DNA...and I use PBS. I try to stay away from optimem as it does contain some protein (~15ug/ml). You should see a fine hazy consistency after you add the fugene to the DNA (appears in a couple minutes or so, especially if you are transfecting ug of DNA) ...and do not remove the media before you add the fugene. Just add it to the media, swirl a few times and that's it. You actually need the media to disperse the DNA around or you will not get an even transfection.
good luck
i doubt it's the DNA. prepared it used qiagen midiprep and nanodrop 280/260 reading is 1.87. but using PBS in place of optimem is new. I'll try this one today hopefully this'll work.
thank you
