Question

Asked 20th Jul, 2011

Christian Medical College Vellore

GC content in DNA!!

Why GC content in DNA sequence is so important? Also its Uses.

Bioinformatics and Computational Biology

21st Jul, 2011

Richard Hall

Luminex Corporation

G-C base-pairs have stronger interactions (than A-T BPs) arising from their ability to form three hydrogen bonds in water. A-T base-pairing yeilds only two hydrogen bonds. That is why the melting point of double stranded DNA is higher for high G-C content DNA as well as for longer pieces of DNA. High GC content DNA can make it difficult to perform PCR amplification, because it is difficult to design a primer that is long enough to provide great specificity, while maintaining a melting point below the optimal temperature of DNA polymerase. DMSO, Glycerol, Salt or MgCl2 can all be used to perturb the effective melting point.

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21st Jul, 2011

Richard Hall

Luminex Corporation

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22nd Jul, 2011

Alia Khan

Islamabad Diagnostic Center

I need exact one step protocol of NS5b pcr.plz help me.email it to me on anila.jahangiri@yahoo.com

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25th Jul, 2011

Akalesh Kumar Verma

COTTON UNIVERSITY

it is site of rich methylation/acetylation that regulate different gene expression

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Is it possible to get good PCR product if GC content of the primer is lower than 40%?

Question

10 answers

Asked 9th Dec, 2018

Hania Ramzan

Is it possible to get good PCR product if GC content of forward & reverse primer is 32% & 35% respectively. Tm of forward and reverse primer is 51.1 & 53.2^o C respectively?

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Question

3 answers

Asked 22nd Mar, 2020

Sicheng Xu

Hello everyone,

These days I've read many papers regarding RNA-seq and its corresponding analysis, I found when it comes to assembly part, almost every paper would tell how many contigs and scaffolds, which are basically called 'unigenes', have formed out of cleaning reads.

Since I'm not familiar with the assembly process I'm looking on the internet and find a pic elucidating the procedure involved which I've attached below. As in the pic, there are four contigs which comprise two scaffolds, name them as 1,2,3 and 4.

MY QUESTION is first why it cannot be the case that contig 1,2,3 or 4 itself rather than larger sequence (scaffolds) they combined would be recognized as 'unigene'.

Second question would be how to determine which two contigs in the pic comprising scaffolds which represent the real transcriptomes, since basically contig 2 and 3 are also able to be gathered as a scaffold, all of them have certain gaps inbetween.

Many thanks in advance.

Melting temperature (Tm) value meaning

Discussion

7 replies

Asked 5th Aug, 2019

Minhsuan Kuo

I have no idea about Tm value in the PRC. I've researched for lots of information but I can't get it completely. Can somebody explain it? Plz

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Question

3 answers

Asked 3rd Apr, 2018

Zoe Evans

I would have thought increasing the temperature would give the reaction more "energy" thus, less specific binding would occur.

However, from my research it seems that if there is unspecific binding (therefore multiple bands on the gel) the solution is to increase the annealing temperature?

Can anyone help?

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Question

11 answers

Asked 30th Dec, 2016

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I used stock BSA 2mg/ml, the standard were 2,1,0.8,0.6,0.4,0.2 in 20µl plus 1 ml diluted dye,I did the standard curve but I am confused did the protein value for the standard would be per ml or per the 20µl standard used, the same problem with the samples I used also 20µl with different dilution So the protein I obtained from the standard curve per ml or per the 20µl sample

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How does a difference in GC content in primers affect PCR?

Question

10 answers

Asked 10th Dec, 2015

Harshraj Subhash Shinde

I am trying to amplify C-DNA by RT-PCR for that I using forward primer with GC content 68.5 and reverse primer with GC content 53. After performing PCR I am not able to amplify my gene. So this difference in GC content between these two primer is responsible for that. Please guide me

Thank you in advance

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23 answers

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GC content in DNA!!

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