What are the factors should consider when designing primers?

More Nilmini Kumari's questions See All

How many restriction sites we could anchored to a primer?

I'm interesting to know that, 1. how many additional nucleotides can you add at the 5' site of the primer ? 2. How many restriction sequences can you add at the 5' site of the primer ? 3. when...

05 June 2018 9,596 6 View

Can RNA contaminate/ interfere plasmid sequencing?

When preparing plasmid samples we do RNAse treatment to remove RNA from plasmid DNA. I would like to know; is it necessary for plasmid sequencing from sanger di deoxy method ? How RNA interfere...

06 July 2017 5,740 7 View

Is this SCI journal " International Journal of Scientific and Engineering Research "?

Please in for me details about this journal. It mentioned it is SCI journal listed in Thomsons Reuters journal list. But I couldn't find it! And please let me know the original web address of SCI...

02 March 2017 7,608 1 View

Can you help me to do ATPase assay using Glo Kinase assay kit(Promega)?

I would like to use above kinase assay kit to check the activity of ATPase in my expressed protein. But I couldn't find a person who has used it in my department. I would like if any body who has...

05 June 2016 9,459 3 View

If the protein contain a his tag, for western blot what sort of Ab is preferred?

My cloned gene contain His-C terminal tag. I would like to check my protein using western blot. So could you please advice me what sort of antibody could be use. I prefered to do the detection...

04 May 2016 8,446 4 View

In colony pcr, why some clones produce less intense bands while some are having very Intense bands in gel electrophoresis?

I use PCR to confirm the insert in my clones. Normally its getting positive or negative bands. But this time I got bands less intense than the positive with primer dimers. some clones only got...

03 April 2016 4,832 17 View

Are there any alternative start codon rather than ATG?

I have a bacterial gene sequence code for a protein but it's start position/ codon is TTG. Is it a mutation or consider as a alternative start codon?. I need to express this protein in a BL 21...

01 February 2016 6,880 6 View

Why 70% ethanol not 100% ?

In labs we are normally using 70% cleaning ethanol for sterilization. Can anybody know the theory behind it?

31 December 2015 747 5 View

Can operon contain more than one promoter?

In bacteria, protein coding genes are organized in an operon. If the operon contain three protein coding regions, My questions are; 1. Do all genes operate from one promoter? 2. Can individual...

10 November 2015 1,807 1 View

What are the usages of N-terminal and C- terminal fusion tags?

pET-28a(+) vector contain few fusion tags. N-terminal His.Tag and N-terminal T7.Tag along with C-terminal His.Tag. What are the individual usage of each tags? If all tags were there with my insert...

09 October 2015 4,782 11 View

How to confirm the site-directed mutagenesis result without performing NGS?

I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...

08 August 2024 4,645 2 View

I can't see the ssDNA band after performing asymmetric PCR. Is there any way to do this?

After performing symmetric PCR, PCR purification was performed. Afterwards, asymmetric PCR was performed using the PCR purification product as a template, but no ssDNA band was confirmed in the...

08 August 2024 1,668 3 View

What precautions should be taken while handling S. aureus enterotoxin Type B in the lab?

I would like to understand potential safety concerns while handling SEB in the lab. Especially while working in animal house facility. Would like to know precautions for handling. Sigma MSDS...

07 August 2024 6,034 3 View

Why after performing site directed mutagenesis ,I don't see any colony after transformation?

I want to introduce a point mutation (change in one nucleotide) into my gene of interest (DNA binding domain) I have designed primers as recommended on the Data sheet of the kit : -Both primers...

05 August 2024 9,059 3 View

Anyone having idea about VN primer for miRNA primer design ?

How to design VN primer to attach with universal reverse primer

05 August 2024 2,116 3 View

How can I inoculate a lyophilized anaerobic bacteria into the culture medium without an anaerobic chamber?

There are anaerobic gas pak jars and environment providers in the laboratory that provide anaerobic environmental conditions for the growth of anaerobic bacteria. However, there is no anaerobic...

05 August 2024 4,945 3 View

How can we now if the promotor in the vector is well?

Hi, everyone. I want to transfer two genes in to the pseudomonas bacteria that isolated from the soil. The bacteria that I want to use for cloning haven't been identified and not be sequenced,...

02 August 2024 3,987 1 View

I'm optimizing a tetra-ARMS PCR protocol with amplicon sizes: 165bp, 123bp and 93bp. Why, in the gel, only 165bp is visible while I'm expecting all 3?

It's an end-point PCR protocol. I'm using 1.5% agarose gel with SyBR Safe dye and TBE as a running buffer, visualization on BioRad XR+ system. I was primarily thinking of primer efficiency,...

01 August 2024 4,673 4 View

PCR showing no bands - Master mix and primers didn't mix?

Hello everyone, I performed a PCR yesterday, and the results showed no bands on the gel. Of course, I probably missed some crucial steps, like adding my samples to the PCR strips themselves, for...

31 July 2024 2,406 6 View

How to retain amplicon yield after Ampure bead cleanup, following a 2-stage PCR protocol?

Hello all, I have been trying to follow a 2-stage PCR protocol used to amplify barcodes of a large yeast library, as per Nyugen et al. (2022) -...

30 July 2024 841 2 View

Ganapati Bhat

Points to be considered for primer design is well documented in Molecular Cloning: A Laboratory Manual. Following links may be helpful to you

https://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/

http://www.nature.com/scitable/content/general-concepts-of-primer-design-8942407

Shami Bakhiet

Length of primer

Target sequence

Sustainability of the primer

Compatability of the primer