6 replies to this topic

[Panoramix]

Hi,

happy to find this forum as I have a big problem with a plasmid I use (actually the most important as my whole project will be based on that  for the next 3 years   )

The problem is that the plasmid seems to be very unstable at least in the conditions I use ! When I transform any strain of e-coli with the original plasmid all the colonies after miniprep and digestion seem to have the correct plasmid. The problem comes when I'm trying to put an insert in the plasmid ! The result I get from every colony is different but none has the correct restriction profile (everyone with different deletion) ! I've tried culturing at 30 and 37, transforming strains like DH5alpha, XL10 Gold, SURE and all give same results ! I 've tried also a second insert ... again with same problems and even worse ... returning from all the minipreps the same plasmid much smaller than the original giving a single band (the original has 5) after the digestion .
(No contaminations)

It seems like my bacteria are playing with my plasmid and my lack with me and my PhD. .....

Edited by Panoramix, 22 February 2005 - 03:00 PM.

[labprincess]

Hiya!

Check your insert and plasmid for restriction sites....you might have some sneeky ones slithering around...mocking you...... Or your enzymes might have some star activity.....in that case they are mocking you...... Re-evaluate your digestion protocol........come on.... you're the eukaryote.....you show'em whose boss!!

Hope that helps
Labprincess

[Panoramix]

Everything is fine with restriction sites !
The problem is with the deletions (only when I put an insert !)

[Sandyme]

Hi,
I am having a similar prob too.. I am using a vector 15kb and trying to clone 3 inserts of 420, 400 and 398 bp each (separately). I have problems in transformation. Just 2 -4 colonies after transformation and the plasmid isolation after miniprep showed a lower size vector than 15.4kb. I suspected the supercoil and performed a single digestion and the vector was smaller size and also i did not get the fall out (bidirectional cloning).. I wonder why my vector is unstable.. The gel extraction seems to work properly and also sfter the CIP treatment..I needs some suggestions..

Thanks in advance.

[allynspear]

I would definitely try to stick with cell lines that are better for unstable plasmids like XL-10 or StBL2/3 cells.  Also, the media that you are growing the cells in will also affect the growth rate, so if you are growing in a super rich medium like 2xYT, the cells may grow too quickly.  I would stick to a high salt LB (Miller's formulation) which is:

10 g NaCl, 10 g Tryptone, 5 g Yeast Extract / Liter of media

I would also recommend plating your cultures in between every liquid passage, i.e. don't passage older liquid cultures to start new cultures.  This is the major cause of minipreps being correct and then maxipreps being wrong.  If you do have a correct miniprep, either re-transform the DNA or re-streak the miniprep culture onto a fresh plate.  When you want to grow a maxi culture, pick a single colony and innoculate 1 mL of LB+Antibiotic in the morning.  At the end of the day (6-8 h growth), add the entire 1 mL of starter culture to your maxi culture (~100-250 mL).  Grow overnight and maxiprep the next day.

The only other advice I can give is to be sure that you know exactly what you are cloning with.  A lot of people will use plasmids that other people have created/modified without ever having sequenced them.  If you don't have a complete verified sequence of your plasmid, I would take the time to sequence the whole thing yourself.  I understand that with a 15 kb plasmid that means probably ~25 seq reactions, but I have discovered many weird things about plasmids I got from other people.  Also, the same goes for your insert.  If your insert is a cut piece from another plasmid, be sure you have verified the sequence yourself.  If your insert is a PCR product, I would try to sequence it directly before cloning.  This is not something I routinely do, but if I'm having problems, I want to be confident in my reagents.  Most sequencing facilities will have a protocol for minimum purification and concentration requirements to sequence PCR products, so I would double check with your sequencing facility to be sure you have enough.

Best of Luck.

[pDNA]

allynspear gave already some good advices!
...but the most interesting thing is why are you plagued by plasmid (structural) instability ...and is there a workaround to prevent this instability?

To shed light on all these things it would be valuable to have some more info on your experiment?
*What is the backbone of your plasmid? ...is your plasmid derived from a commercial vector? ...please provide any information on the plasmid (antibiotic, origin of replication, promoters and so on)!

*What is the purpose of your vector ...do you need it for expression of a protein in bacteria/yeast/mammalian cells? ...do you clone into a retroviral system? ...what are you going to do after sucessful cloning?

*What type of insert are you cloning? ...and for what reason?

Maybe if you provide more information it get easier to help you (at the moment your question is like: "Doctor, i'm somehow sick ...what should i do?")!

Regards,
p

[pDNA]

...and i forgot to comment on allynspear's tips:
also consider good aeration for all plasmidpreps!!! ...problems often start when cultures are have poor aeration! ...set you shaker at high speed (250-300 rpm ...depends on the excentrity) and use only a 1/5-1/4 of the volume to provide enough head space in your vessel.

Regards,
p