Restriction enzyme cutting site in pRARE plasmid .

08 August 2017 1 1K Report

Dear All,

Does anyone know the sequence of p-RARE plasmid or any unique restriction enzyme cut site just to confirm that the plasmid I have isolated is a right one? I have gone through few publications and it says pARE has a Aval cut site, but ,unfortunately, we do not have any Aval in our lab. Any advice?

Thanks in Advance

Shouguang Huang

I tried to search this plasmid in NCBI database, but I did not find it....

Badges
Science topic

More Abir Chakraborty's questions See All

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

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

How can I edit suggested publications?

Hi there! I regularly receive suggestion about papers and authors that are totally out of my area. Does anybody know how I can fix this? Thank you!

01 March 2021 9,574 4 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

When is it unnecessary to change organisational culture?

As a former management practitioner, I recall a generally accepted view that changing organisational culture is difficult, so you should only attempt it if the organisational change that you are...

27 February 2021 2,595 8 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