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

Can I use SLA1 instead of XHO1?

Hi there. I want to know is it possible to use SLA1 Enz instead of XHO1? My gene of interest is CD20 And I'm working on PSpax, PMD2G plasmid and PCDH. Any suggestion or comment would be...

08 September 2018 8,697 3 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

Protein-aptamer gel electrophoresis help, please??

Hi, I have problems with running gel electrophoresis. I have tried agarose gel electrophoresis and native PAGE. I have two proteins, which have molecular weights of ~30kDa and ~180kDa and two...

03 March 2021 4,275 4 View

Can linear DNA be used as template for site-directed mutagenesis?

Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...

03 March 2021 401 4 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 View

How to regulate SSCP temperature in simple PAGE?

I'd like to perform single-strand conformation polymorphism (SSCP) in my thesis, however I cannot control the temperature of the vertical PAGE since we are using the conventional tanks. Is there a...

02 March 2021 9,157 1 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 are my bands weird in this Agarose gel?

Can someone please give me some possible things that could go wrong? Here is my recipe: 0.5g Agarose 50 mL of TAE 1x 1 uL ethyl bromide. Gel was run at 100V for 1 hour. The buffer used is also TAE.

01 March 2021 9,952 3 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

Why my agarose gel staining sometimes looks not homogenic but like gradient (see the figure)?

I am worried about this overexposure of the upper part of the gel in the picture. Is it possible that this is the result of too much concentration of ethidium bromide? Why is there such a big...

25 February 2021 8,140 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

Difficulty in cloning of 1kb gene into vector?

I am facing difficulties in cloning a 1kb gene into a vector (pJIT163). I have my gene on interest (GOI) in pUC57 and want to clone in pJIT163 using SalI and BamHI restriction sites. I am getting...

25 February 2021 3,221 7 View