Toxicity of Lipofectamine 3000?

Dear all, I have been struggling with transfection in Hs578t with my pure plasmid ( maxi prep plasmid). I transfected in a 24 well plate , changed the media after 6 hours post transfection. I...

08 September 2019 6,186 1 View

Max extension time for pcr with Phusion Hot start II DNA polymerase?

Hi all, I am planning to PCR a 7430 bp fragment from a big plasmid(11 kb) with Thermo Phusion hot start II DNA pol. Thermo recommend to use 15s-30s as extension time per 1 kb length. Overall GC%...

07 August 2019 2,980 3 View

Is it very hard to transfect Hs578T cell ?

I am planning to transfect Hs578T cell with pcDNA eGFP. Is it very hard to transfect? We have Extreme gene HP transfection reagent. Any advice from people if they have done that before?

06 July 2019 5,737 2 View

How efficient and good is NEB Q5 Site Directed Mutagenesis kit?

Hello All, I just want to know your thoughts on NEB Q5 site directed Mutagenesis kit. Has anyone used this ? Is it good ? I have a plasmid of 6 kb on which I design my SDM . Your advice and...

05 June 2019 9,287 6 View

What is the maximum insert length limit for ligation?

Hi All, I am planning to clone one of my inserts into VC155 BiFC plasmid. My insert size is 8 kb where vector size is 5kb. Is it very hard to clone 8 kb into 5 kb? Also I would like to do...

04 May 2019 6,586 4 View

Why do we need at least 1 pmoles of DNA for Alkaline phosphatase mediated dephosphorylation?

I have been struggling with a cloning and its due to the fact that It is digested only with a single restriction enzyme(I have no other option). I was wondering why do we need 1 pmoles of DNA for...

03 April 2019 2,416 10 View

What is the alternative way to clean Ni-NTA column other than GuHCL?

Hi All, I need to wash and recharge Ni-NTA column. Unfortunately, I do not have GuHCL now. Can we use 8M Urea instead of GuHCL? Is it also necessary to wash recharged beads with Urea+ 0.2 M...

02 March 2019 4,485 3 View

His tag protein purification at Room Temp?

Dear All, Can we incubate Supernatant and Ni charged beads for binding at room temperature for overnight? Currently, we do not have a fixed temp room in our building. What about keeping Ni...

01 February 2019 6,671 6 View

Impact of N terminal His tag on signal sequence of secretory protein?

Hi All, I recently generated few constructs in pcDNA 3.1 HisC,encoding three different Fibronectin fragments.I did not realised that His tag was at the N terminal(I just overlooked). I...

10 November 2018 2,354 1 View

Can we store pierce glutathione agarose beads in 50 mM tris, 150 mM NaCl,pH 8.o ?

Can we store pierce glutathione agarose beads in 50 mM tris, 150 mM NaCl,pH 8.o after regeneration?

10 November 2018 6,963 3 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

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

Standard curve shifting over time. How do I stabilise the ELISA?

24 February 2021 1,365 3 View

How to get the vector of a specific vibration mode in phonon dispersion?

For example, I have this phonon dispersion(Fig. 1), and now I would like get vector of atoms which describes TA1 in P. I know v_sim can show vibrational animation of specific mode, but I do not...

20 February 2021 4,332 4 View

Specify the Burgers vectors for dislocations, which cannot be uniquely identified.?

1. A bcc metal single crystal was sliced along (1 -1 0) plane for TEM study. Assuming that the TEM available is equipped with a goniometer stage which tilts up to 45o. The Burgers vector of...

20 February 2021 2,370 1 View

I am working on image transmission, i converted the image into binary vector, i want to extract the MSB of each pixel ?

i have a vector 1*512 size, i want to take the first value every 8 elements and put these elements at the end of the vector,how to do this on matlab ?

19 February 2021 2,352 5 View

Sanger Sequencing results not matching experimental results of Restriction Digestion, colony PCR and protein expression?

I have cloned two domains of a gene into two bacterial vectors using cut and paste cloning with different RE sites. I checked the putative clones by colony PCR and Restriction Digestion (fallouts...

18 February 2021 6,736 4 View

Does the property of GFP affect a fusion protein's localization?

Hello, I am planning to insert GOI between CaMV 35 promoter and mGFP in pCambia1302 to produce a fusion protein and see subcellular localization of the protein. But the vector says it's membrane...

15 February 2021 4,353 3 View

How much amount of DNA (ligation mix) should I use for transformation?

Hi Fellow Scientists! I am trying to clone my 300 bp insert into a 6 kb vector. I run a few different ligation reactions using (1:3 vector: insert molar ratios) 10, 20,30, and 40 ng/ul vector...

15 February 2021 9,563 6 View

Can I express protein of plant origin in E. coli strain (M15) using pET28a vector systems? Are M15 strains and pET vector systems compatible?

Are E.coli strains M15 and pET28a vector systems compatible? Can I over-express my protein which is of plant origin. Previously I was using BL21 DE3 strains. Was confused to use M15 strains for...

10 February 2021 2,564 3 View