Why am I getting 2 bands of 70 KD and 55 KD for the chains of my antibodies on a SDS-PAGE gel?

Dear All, I have purified 3 variants of the monoclonal antibody from CHO cell line expressing them, using 30 KD amicon ultra-4 centrifugal filter devices following by protein G purification,...

06 July 2019 7,989 13 View

Is it practical to repeat infection process to reach better transduction efficiency?

Dear all, I'm working with lentiviral vectors and aiming to overexpress my gene of interest in CHO-k1 cell lines. I have tried several protocols to reach the high titre of the virus, however the...

04 May 2019 852 4 View

Plasmid transfection in HEK293T cell by Calsium Phosphate method ?

Dear All , we are working on gene transfection by Calcium Phosphate method but after 24h when I want to change the cell culture medium of my test and contro cells , I see that tests are not as...

11 December 2018 3,646 8 View

Which CD20 expressing cell line is better?

Hi there , we are working on antibodies against CD20 antigen ( tositumomab, ofatumumab, Obinutuzumab ) and need a cell line which overexpressing CD20 . as far as I am concerned, Daudi , Ramos...

10 November 2018 7,058 5 View

What is the best protocol for lentivirus transfection in HEK293Tcells by "CaHpo4" ?

Hello everyone , I'm trying to do transfection by lentiviral vectors and my packaging cell line is HEK293T. We are going to do calcium phosphate method.I would like to find the best protocol for...

10 November 2018 3,973 4 View

Generating Stable Cell Lines with Lentivirus ?

Hello everyone We are working with HEK293T cell line. I would like to know which is the best protocol to Generating Stable Cell Lines with Lentivirus(2nd generation) ? any suggestion or comment...

09 October 2018 3,551 2 View

PEI(polyethylenimine) versus CaHpo4 and lipophectamine transfection for virus production in HEK293T cells ?

Hi, I was just wondering if anyone has any personal experience or opinions on using PEI for the production of lentivirus in 293T cells compared to calcium phosphate precipitation and...

09 October 2018 2,286 10 View

Any suggestion or comment in regard of PCR troubleshooting ?

07 August 2018 6,772 11 View

PCR troubleshooting.what should I do to solve the problem ?

07 August 2018 4,152 7 View

What is the problem when we can not see anything on agarose jel electrophoresis ?

I'm trying to clone my specific gene , My sample is 900bp cDNA,after pcr i run it in 1% agarose jel, 100v But I couldn't see any band of marker as well as sample. Any comment and suggestion in...

07 August 2018 5,098 4 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