How to confirm I have a full natural plasmid?

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What Contaminates Cause Loss of Resolution in Plasmid Sanger DNA sequencing?

We use an AB 3130xl Genetic Analyzer for Sanger Sequencing, and on occasion, we see our plasmid samples struggle with wavy, loss of resolution (as seen in the attached image). Our general...

14 January 2021 7,346 6 View

Disappearing Coomassie Bands?

Suddenly my Coomassie staining has produced strange results (see photo attached). The past 8 gels all share the same trends: the bottom half of the gel fades out and is absent of any bands. I...

19 December 2020 3,619 1 View

What is the maximum passage number of SKOV-3 and SKOV-3-Luc cells?

Hello, for my experiment I'm working with SKOV-3-Luc cells, which are SKOV-3 cells stably/continuously expressing the firefly luciferase enzyme. These cells are obtained by the transfection of...

25 November 2020 3,219 3 View

Masters thesis subject - International humanitarian law/ International criminal law?

Hello I am a master's student and I'm searching for a current debate in the field of international humanitarian law or international criminal law. Is there somebody who can help me with this?...

08 November 2020 1,571 13 View

Is it possible that a IncX and IncF plasmid fused?

I recently performed plasmid extraction and nanopore sequencing. I have found what looks like a plasmid with replicon types IncX and IncF. Even with spAdes, canu, unicycler (illumina and nanopore...

29 September 2020 7,778 2 View

Any suggestions for a simple method to reduce the viscosity of bacterial growth media?

Hi, I am working with a bacteria from the genus Paenibacillus. I am having trouble separating it from the media (PDB), and difficulties in extraction of secondary metabolites from the media,...

04 August 2020 9,974 3 View

Can I make a primerset (for RT-qPCR) for one single transcript variant? If yes, how?

I am trying to perform a RT-qPCR and I would like to design a primerset for one single transcript variant for RT-qPCR. The problem is that it is very difficult to get primers that only bind on one...

22 April 2020 3,071 4 View

Can anyone tell me which species (or higher taxon) this is?

We found this small, maybe parasitic, crustacean in the North Sea offshore. The posterior body part is missing, but it looks quite characteristic. There is double-hooked rostrum, 2 antennae...

08 April 2020 6,842 9 View

Can I use time as a predictor variable in a panel regression?

Is it possible to generate a new variable "phase" that is based on the time the participants were questioned? I generated this new variable by dividing the time into three phases (beginning,...

30 March 2020 3,756 6 View

Dielectric constants of organic:aqueous mixtures, is linear addition a valid approximation?

I would like to compute dielectric constants of organic:aqueous mixtures. Would linear addition of the dielectric constants of the pure solvents give reliable results? For example, for a volume...

26 March 2020 5,939 3 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

PUC19 as a transfer plasmid for lentiviral delivery?

Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...

28 February 2021 4,868 2 View

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 3 View

How can we use a hybrid element in Abaqus Dynamic/Explicit for impressible materials?

Hi, all! How can we use a hybrid element in Abaqus Dynamic/Explicit for impressible materials? I have not find the choice for explicit element in Abaqus CAE. I am wondering whether it is possible...

25 February 2021 9,936 2 View

How do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?

I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...

25 February 2021 6,635 5 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View

Frederic Lepretre

there are lot of aligner, even on web, but you can use this one from EBI:

https://www.ebi.ac.uk/Tools/msa/muscle/

all the best

fred

Vuong le

https://cge.food.dtu.dk/services/PlasmidFinder/

You can try PlasmidFinder for plasmid typing.

Alternatively, you can annotate the plasmid and check for featured genes like conjugative encoding genes and plasmid replication gene.