I have a MISSION shRNA plasmid DNA provided by Sigma (please see the information in the attched file).
I transformed the plasmid into TOP10 Ecoli strain by heat-shock method. After growing on agar plate with ampicillin O/N, I got the colonies (much smaller than I got from other plasmid DNA e.g. TOPO vector or pGL3 vector). I picked up 3-4 colonies and innoculated in 3ml of LB medium O/N, the OD of innoculation is much lower than I got form other vectors. I used 1ml of the innoculation for miniprep and I got the plasmid DNA on the agarose gel (low concentration). Then I used 200ul of innoculation grown in 200ml LB mediums for maxiprep and I got... no pellet and no plasmid DNA...
(I did the experiments of the shRNA plasmid DNA concomitantly with pMISSION VSV-G plasmid DNA and pMISSION GAG POL plasmid DNA, these two plasmids gave me a high DNA yield)
The problems are:
1. Small colonies
2. Low OD after innoculation
3. No plasmid DNA in maxiprep.
Can you suggest me the reasons and how to solve them?
Thank you very much!
Hi, Duy,
I have some questions.
Did you transform your plasmid and other plasmids at the same time to the same type of competent cells? If yes, whether they have same AmpR? Small colonies make me think about satellite colonies. Ampicilline plate usually has this problem. If possible, change to carbenicillin or make new Amp plate.
Make sure you didn't make any contamination when handling.
If you try one more time and still same problem, I suggest to change competent cells (DH5a, JM109).
Thanks Dr.Chuong,
Nice to hear from you. Hope that you are going well ^.^
Thank you for your suggestion. I performed the experiments and used the agar plates which were prepared in the same time. I tried to prepare new Amp and made new agar plates but the problem was not solved.
I think they were not satellite colonies because there were some smaller colonies around them and I did not pick up those satellites. However, somehow the colonies may be contaminated by the satellites when I picked up them.
As your suggestion, I will use carbenicillin instead of Amp and use DH5a strain instead of TOP10.
Thank u again and warm regards ^.^
Hi Duy,
Low yield suggests that you are dealing with a plasmid that is toxic for bacteria or not replicating well. This is further supported by the fact that in the 200 ml culture you got no pellet. You can grow you bacteria at 30 degrees instead of 37. This will allow more time for proper bacterial growth and plasmid replication.
Hi Duy
I am trying to generate the two MISSION packaging vectors pMISSION VSV-G and pMISSION GAG POL. I've looked at the vector maps but I don't see an antibiotic resistance gene in the smaller pMISSION VSV-G vector. Could you let me know how you generated it?
Best wishes,
Emma
Hi Emma,
I actually received those plasmids from my senior and he left our lab. I do not know whether that plasmid (VSV-G) is pMISSION one or not. However, screening with ampicillin works well for VSV-G plasmid.
I think you may want to send Sigma an email and ask for that issue. They may give you an appropriate explanation.
Good luck
All the best,
Duy.
Thank you so much for your reply. I have emailed Sigma and they are speaking with their people in the U.S.A, so hopefully I'll get a reply soon. I'm giving it a go anyway and will try using a diagnostic digest to identify the product.
Best wishes,
Emma
Hi Emma,
If you have their answer, please let me know. It may be helpful for me for my future work.
Thank you so much and good luck always,
Duy
Hi Duy
I haven't heard back from Sigma, but I ran a sequence alignment and the VSV-G vector does contain the beta lactamase gene, so that makes life easier!
Best wishes,
Emma
Hi Emma,
Great to hear that and thank you for your information.
Best luck to your project :)
Duy
Dear Duy, I'm having similar trouble. I'm not being able to get high concentration DNA (VsVg and Delta 8.9) from the overnight bacterial growth. How did you deal with your problem?
Use a strain for transformation suited for lentiviral plasmids, like Stbl3, NEB stable, Lucigen Endura.
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