How can I analysis different RNA fragments processed by in a solution?

Hi everyone, I am trying to perform a in vitro DICER assay to see if the RNAs that I am studying could be cleavage in to small RNA fragment. There might be many different fragments in my total RNA...

10 November 2018 3,532 2 View

How can I improve the low yield of gel extraction by GenepHlow Gel/PCR kit?

08 September 2017 6,872 7 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

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

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

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

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

Why are Sanger Sequencing reads interrupted in some edited clones?

I have a Crispr/Cas9 edited bulk population and I can briefly check the indel ratios in the bulk population using Sanger sequencing. After cloning single cells, I wanted to check the indel types...

18 February 2021 8,198 6 View

Hi, can anybody help me with the suggestions how can I amplify a stress induced gene of 1107 bp?

I am trying to amplify the TGA1 (Transcription factor 1 ) of Arabidopsis ,which is 1107 bp. I have treated the plants with Salycylic solution over night and extracted the RNA and make cDNA. I...

17 February 2021 7,802 2 View

Alternatives to traditional cloning of insert into backbone (dsDNA)?

Hi, I am stuck with a cloning experiment: The insert & backbone(bb) share one compatible end at 3' end(NotI) and one incompatible end at 5' end (XbaI for insert; HindIII for backbone). So i...

16 February 2021 1,664 3 View

Why do I have trouble with cloning duplicate gene in the same plasmid?

Hello! I am trying to generate a construct in this format: seqA - seqB - linker - seqC - seqB into pSF-CMV-Ub-Puro-Ascl plasmid 0.5kb 2kb 66 0.25kb 2kb I have tried many...

15 February 2021 7,118 3 View

Would using an asRNA for repressing transcription of a high expression Listeria monocytogenes non-coding RNA harbored in a plasmid be a viable option?

Hello all, I am interested in deleting a highly expressed non-coding RNA located on a Listeria monocytogenes plasmid. Currently, there are not any optimized gene deletion methods for Listeria...

09 February 2021 4,994 1 View