Adeno-X Expression System problems - (Oct/13/2005 )
Hi there!
I was just wondering if anyone has experienced problems with the Adeno-X Expression System 1 from BD.
I have cloned my GOI into pShuttle2. The problem I am experiencing is cloning my GOI from pShuttle2 into the Adeno-X DNA using the unique restriction endonucleases I-CeuI and PI-SceI. The issue is that I obtain colonies upon transformation of XL-10s with my GOI in the Adeno-X however I observe no growth when transferring colonies to liquid culture. I have performed extensive optimisation of conditions that include altering incubation times (>72hours) and temperature (30-37 degrees C), antibiotic resistance levels (10-100ug/ml ampicillin), selection of different size colonies, spending many months troubleshooting without success. I set up a control whereby I transformed XL-10s with adeno-X DNA alone which produced no colonies as expected, suggesting I am not obtaining null vector containing colonies.
I was wondering if anyone has experienced similar problems or has any suggestions on why the colonies grow without problem on plates but show no growth in liquid culture. I would appreciate any help on resolving this problem.
Many Thanks,
Debra
I was just wondering if anyone has experienced problems with the Adeno-X Expression System 1 from BD.
I have cloned my GOI into pShuttle2. The problem I am experiencing is cloning my GOI from pShuttle2 into the Adeno-X DNA using the unique restriction endonucleases I-CeuI and PI-SceI. The issue is that I obtain colonies upon transformation of XL-10s with my GOI in the Adeno-X however I observe no growth when transferring colonies to liquid culture. I have performed extensive optimisation of conditions that include altering incubation times (>72hours) and temperature (30-37 degrees C), antibiotic resistance levels (10-100ug/ml ampicillin), selection of different size colonies, spending many months troubleshooting without success. I set up a control whereby I transformed XL-10s with adeno-X DNA alone which produced no colonies as expected, suggesting I am not obtaining null vector containing colonies.
I was wondering if anyone has experienced similar problems or has any suggestions on why the colonies grow without problem on plates but show no growth in liquid culture. I would appreciate any help on resolving this problem.
Many Thanks,
Debra
unfortunately, I met the same problem recently. The reason I think may be due to the toxic of plasmid which kills the bacteria or the unstablility of the plasmid (in my case, the size of plasimd is around 12 Kb, maybe too large?). Do you try to use the other kind of competent cell? eg. ABLC suitable for toxic clone (invitrogen).
Do you solve your problem?? if you do, please mind me know.
ok guys
i'll tell you my story
i spent 1 year on that AD-X
sign
may be because i'm a stupid post doc with bad hands... i don't know 
BUT
i finally got that adenoviral dna
so what i did
after digestion with that unique enzymes and ligation with AD-X vector (that should be done at 16C degrees) AND! digestion with SwaI enzyme (you have to do all steps exactly as in protocol) I transformed vector into E.coli with OneShot Top10 from Invitrogen. I tried to do the same with DH5a but it didn't work very well for me. After I got colonies (which are very small and if you will get a LOT of them it would mean you SwaI digestion didn't work) I went with PCR checking. Then I grew positive colonies (which had band with size around 300bp) in 3ml of LB with Amp 100ug/ml for 7-8hrs. I was getting from 7 to 14 positive colonies because sometimes some of them didn't grow. But that which was growing I transformed to 200-300 ml of LB+Amp for o/n. Then I purified DNA with Endofree Maxi kit from QIAgen. That kit was recommended to me after I failed to get any DNA with mini, midi, maxi preps.
Last thing. E.coli after transformation is very sensetive and will not grow in LB media after 2 wks or even less 
. So you always should work with "FRESH" colonies. Do not keep plates for more than 1 week.
Now I'm trying to distinguish CPE and cell necrosis in overgrown cells . They say I have to get CPE in 7 days. It means cells will be overgrown...
Thank you for your replies.
I tried using the Adeno-X kit for over 6 months without success so I stopped trying! Thank you for your comments, I will probably go back and see if I can get the kit to work.
Debra