Here are 3 other suggestions-
Bacteria may mess with DNA, especially the AT-rich Dicty DNA, if they are stressed, so in addition to growing bacteria with Dicty inserts at room temp-30 degrees as Thierry wrote,
1) put 1 ml of culture into a ~2.5 cm diameter test tube, and have the tube shaking in a slanted position to maximize surface area and gas exchange
2) grow these cultures up to a 'heavy smoky' density, not to an 'opaque paint' density
3) on plates, pick the colonies when they are pinpoints, don't let the colony get larger since this stresses cells at the center of the colony (accumulated waste products..)
cheers
Richard Gomer
________________________________________
From: DICTY [[log in to unmask]] on behalf of Kari Naylor [[log in to unmask]]
Sent: Tuesday, August 31, 2021 10:54 AM
To: [log in to unmask]
Subject: Re: [DICTY] pDM323 and pDM317
Thank you so much, everyone, for all the great info (got some personal emails too). I am very glad to hear that it's not just me. I don't have the money for cloning as I do so little of it that everything expires before it's used up thus I usually farm out my stuff. I thought maybe I just wasn't doing my midipreps correctly. pDM320 was easy to work with, so these GFP vectors threw me for a loop. They transform so much more efficiently into AX4 than the older ones that I really want to keep working with them. New plan, use XL10 golds and if anything is hinky, go back to original stock. Thank you again
Kari Naylor PhD
University of Central Arkansas
Biology Department
180 Lewis Science Center
ph:501-450-5826
[log in to unmask]<mailto:[log in to unmask]>
On Tue, Aug 31, 2021 at 7:47 AM Thierry Soldati <[log in to unmask]<mailto:[log in to unmask]>> wrote:
Dear Kari,
I cannot really comment on the specific problems you have now, but maybe share a warning about the vast pDM series of vectors.
These vectors have been instrumental in the last decade of work in my group, and the recent extension to the collection by Peggy Paschke opens new avenues.
Despite this, I must admit that we have encountered, from time to time, in an unexplainable manner, some serious problems of rearrangement. This can obviously be due to some of the redundant use of vector parts (promoters, terminators etc..), but again, we did not really find a rational explanation to discriminate vectors that rearrange from the ones that don’t.
The precautions are to use the right E. coli strain, and we have had best luck with XL10-Gold (which were designed to accept large genomic libraries) and DH5alpha.
The second one is to grow the bacteria at low temperature (somewhere between RT and 30°C), and with high ampicillin (or the more stable carbenicillin).
Also, sometime using a “low copy vector” kit for the DNA purification helps.
When you run an agarose gel, always use precise size-markers and watch for multiple bands and shorter vectors that result from the rearrangements.
In case of doubt, make a careful restriction map before use.
Any shadow band, and any deviation from the map or sequence should be a definite NO-GO signal.
Good luck,
Thierry
On 30 Aug 2021, at 22:09, Kari Naylor <[log in to unmask]<mailto:[log in to unmask]>> wrote:
Folks I usually have Genewiz do my cloning and they are having a heck of a time with these plasmids
1) they couldn't get any sequence from them, I and DictyStock Center confirmed by digest that the plasmids are correct, and a new primer was suggested
2) now they are unable to transform them into DH5alpa cells. I plan to go and test this myself but that seems a bit weird to me.
3) Finally they were able to get sequence from pDM323 and found a couple of insertions
Vector pDM323 was sequence-verified and two insertions were noted upstream of the 5’ BglII site.
[X]
I have attached a word document with these insertions. I haven't not looked closely yet to determine if these insertions would cause trouble but basically I am writing to see if anyone else has this much trouble with these vectors and if anyone knows about these insertions.
Thank you in advance
Kari
Kari Naylor PhD
University of Central Arkansas
Biology Department
180 Lewis Science Center
ph:501-450-5826
[log in to unmask]<mailto:[log in to unmask]>
<image001.png><Sequence verification of vector pDM323 .docx>
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Prof. Thierry Soldati
Department of Biochemistry
Faculty of Science
University of Geneva
30 quai Ernest Ansermet, Sciences II
CH-1211-Genève-4, Switzerland
Tel: +41-22-379-6496
email: [log in to unmask]<mailto:[log in to unmask]>
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@SoldatiLab
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