I bought a synthetic gene from a vendor, the gene is ligated in the pUC57 plasmid (with ampicilin resistance). the pUC57 plasmid have been tested using electrophoresis and it's all good.
I'm trying to transform it to the DH5 cells to store and propagate the plasmid, but it's failed (it's been 6 months trying). i have been using some methods such as CaCl2 and SMOBIO CK1000 transformation kit, heatshock. and using ampicillin LB plate (100 ug/ml and recently i use 200 ug/ml)
i have specific primer to detect the gene inside pUC57 plasmid, the colony i pick is shows positive result (PCR) but when i isolate the plasmid (from liquid culture using GeneJet Plasmid MiniPrep kit) and run it in agarose gel electrophoresis it shows smear (really thin) i also run it with positive control (also DH5 that have a known plasmid in it, so i think this could ommit the possibility of dnase contamination in the plasmid isolation kit).
does anyone have ever encountered something like this? or anyone have any advice? thankyou in advance:)
Hi Jonathan,
If I understood correctly - you bought the pUC57 plasmid with your gene in it, transformed it into DH5alpha, got normal colonies, confirmed the presence of your gene using colony PCR, but when you inoculate into liquid culture for plasmid isolation you get no plasmid (or the faint smear)?
How does your plate look after transformation? Do you get an expected amount of colonies (e.g. well separated colonies after plating cca 20 ul from 1 ml of recovered bacteria after transformation)? Do you get colonies after overnight incubation or do you prolong the incubation time? When you inoculate a single colony for plasmid isolation what is the OD in the morning - to me it sounds like you are isolating plasmid from colonies that are devoid of a plasmid? I assume your plasmid isolation procedure is OK, since you said you isolated another plasmid in parallel and the yields are ok.
Your Amp concentrations are a bit high (the second one in ug/ul is I assume a typo), and the stringency of selection might be higher in liquid culture.
I would also send the original plasmid for sequencing with various primers, not just for insert (would focus on the resistance gene and ori regions just to make sure everything is as specified).
Ana Crnkovic Yes that;s right.
my plate looks like perfectly ok, i didn't count it precisely but i believe there is more than 70 on the plate.
and i get the colonies after around 14 to 16 hours incubation at 37C.
and the liquid culture is incubated for around 6 hours at 37C before isolating the plasmid
Yes, sorry for the typo, it should be 200ug/ml..
Jonathan Jonathan 6 hours for liquid culture is quite short, why not use 16 hrs (overnight) to allow bacteria to replicate more plasmid? also, have you tried transforming a new batch of bacteria with the "smeary" plasmid you have? even if there is a little bit of plasmid, your second transformation might work (this would then indicate that it is there just not in very high amounts).
Ana Crnkovic yes it's quite short, usually i do 18-24 hrs of incubation for plasmid isolation. but theoretically pET should have lower copy number (15~20) than pUC (500~700) so i think that should not be a problem if i got the pET plasmid quite ok..
Thanks for the suggestion, i will try to transform new batch with the "smeary" plasmid..
You mentioned that the colony PCR is working but you have problem with Liquid culture. I would say that 6 hours is too short. An overnight liquid culture att 37C would be worth trying.
Naveed Asghar thanks for the advice, I will try it tommorow and give an update saturday/sunday
Ana Crnkovic Naveed Asghar
i attached the gel doc of plasmid isolate today..
Lane 1: nothing, Lane 2-4: plasmid isolate, Lane 5: 1kb bp ladder, Lane 6-7 : plasmid isolate. the culture incubated for 16 hours with 100 ug/ml ampicilin (37°C).. any suggestions?
Jonathan Jonathan
I do not see the attached picture, but I assume the isolation did not work out again? I would definitely then send the plasmid for sequencing (with multiple primers).
I'm sorry, here is the attachment.
the plasmid is already sequenced before sent to us, also my primer is already specific for the gene.. i really can't figure out whats wrong with this..
Is the cell mass from the liquid culture comparable to what you get with your other Amp resistant plasmids? Did you recently isolate some other plasmid with the same kit (i.e. you are sure that all buffers are OK and, for instance, EtOH has been added to the wash buffer)?
Yes, the cell mass from pUC57 is more than pET-20b
I'm using the same kit for isolating the pET 20 b and the result is perfectly ok
Could it still be stuck on the matrix? Have you tried loading the supernatant after you neutralize and centrifuge the sample (prior loading on the column)?
i have not.. i will try this tomorrow. Thank you, i will give an update after that
nothing changed..
i believe the one thing that wrong is my cells, but i don't know how to fix this
Is your DH5alpha stock is stored properly? If you still has the plasmid stock you can try transform it again using a freshly made competent E. coli to see if there is a problem with your E. coli
You could also try isolating plasmid using alkaline lysis method without using kit.
Dear All,
The problem is fixed; it's caused by plasmid aggregation I guess
because when I try to linearize, it somehow shows a single band with the correct size in the gel.
Dear Janett Torr ,
since there is high sequence homology between plasmid, it might be plasmid aggregating with each other in that homologues. I'm not really sure about that but that's things on my mind
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