Copy Number Determination Optimisation Using Real Time PCR Machine

The objectives of this study is to  check what gene is suitable for creation of a standard,to establish real time PCR as a high throughput technique for copy number work and to optimize / prepare a good standard for absolute quantification analysis for this work.

We designed two strategies for the optimization of qRT-PCR. The first one is through Southern blot copy number correlation with qRT-PCR. The second is through the creation of standard using a plasmid carrying the construct that was used in the plant.

In lieu of the first objective to find a gene suitable for doing copy number work we encountered problem with NOS gene. 

My Work As a Molecular Biologist

by AP

My workbench where I did primer design, computations,read  literatures and of course analysis.....

Multiplex PCR the one I've missed to use

For confirmation of primer design( checking for size amplicon size and troubleshooting)

My baby:  Roche qRT-PCR  where I did quantitative gene expression analysis 

RNA room: RNA extraction, PCR  stock preparations, cDNA synthesis and normalisation. my dwelling place.haha

The sight outside of the RNA room of our laboratory....

Fossil Hunt and Naturalist Expedition

Palawan, Philippines- I am in Palawan this week to collect fossils in a nearby Batang batang River, the same river where I used to swim when I was a kid. I will also collect pictures of indigenous plants and animals in Palawan. I will post them in this blog next week.

Human Instinct

Beauty is defined by symmetry. Most of us adhere to that general concept. When looking for a potential mate my female friends prefer a handsome looking guy or an intelligent guy. The same goes with my male friends except that they are more critical  with the physical appearance. It is an animal instinct to look for a more superior trait in a potential partner to ensure reproductive success and be part of the ongoing evolution.

Phenotype or the physical appearance of an individual is dictated by what is encoded in the DNA sequence. Human being can sense good traits from a potential partner even at the first few seconds of encounter. We can gauge or seize up the person if he or she can be your partner or lover. It is indeed a human instinct.

Summer Professional Researcher

I've worked as a summer researcher of the Tissue Culture and Biotechnology of the International Rice Research Institute(April 15-May 30,2010). I worked again with iron transporter genes in rice. The experiment went successful.

Detailed Steps on Primer Design

Detailed Steps on Primer Design
I list here the steps on how to design a good working primer.
1. Get the sequence of your gene from published literature for example OsNAS3.
2. Using the TIGR Blast/ NCBI Blast, check the whole sequence if it specifies the gene of interest. It is important that the gene sequence is 100% homologous to the plant where the sequence is derived and the most important is that the whole sequence specifies only one distinct gene.
3. Put your sequence inside the box of Primer 3 online software. Specify in the software what are the parameters of the primer you want to have. Click generate. You would get something like this.
Important Notes:
When designing primers take note of the following:
GC content, Tm o primer, 3’ ends of the primer
PCR product size- It depends on what your purpose is. If your going to used the conventional PCR you can create a primer with a product size of 400bp and up. If you’re going to use the primer for real time PCR design a primers with 100-350 size only.
4. To be extra sure if your primer is really specific, Blast them again with NCBI Rice Blast. This would look like this. With the use of Vector NTI/Bioedit you can check the size of your PCR product.
5. You are now ready to order your primers at SBS or proligo.

Gene Expression Results

I am getting various trends in gene expression of the genes I am studying. Some genes tend to be express early on the development while others were highly expressed on the later stage