Commercial IL12 expressing plasmid?

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Have you ever had any problem to confirm cloning by restriction analysis?

I have made a construct including 2 PCR produced inserts and a CMV-EGFP-bGH poly A between them. At first, insert 1 was created by PCR and cloned into pSL1180. Cloning was confirmed by...

02 March 2018 8,476 2 View

Does anyone have any kind of experience with viral homologous recombination?

Hi I am using Homologous Recombination to make change in my virus DNA with the plasmid construct which I have already prepared . I have heard this method is really hard and time consuming to be...

02 March 2018 9,875 7 View

Why pET 21 should be reversed for displaying in software such as CLC or Snapgene?

I am trying to insert my fragment in pET21 plasmid, but if I do not reverse pET21 plasmid in CLC or other software, the direction of restriction sites will be disrupted. I was wondering if anyone...

31 December 2017 5,505 4 View

Does any one know how I can find sequence of pAdenovator-CMV5?

Could you please introduce me a database to find this plasmid sequence? I could not find it in Addgene and any where else that I know. Thanks

31 December 2017 1,551 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 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

Best plasmid could be used as a standard curve for AAV titration?

I have been trying to have a good AAV titration for my production and I have tried different plasmids to set up as a standard curve but each one behaves differently. i am trying to use ITR for...

23 February 2021 5,167 3 View