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

Beach in Southern Palawan

Here is a video of a beach I took from Narra, Palawan. It portrays the rich mangrove forest of Narra facing the South China Sea. On the background includes the fire we set up to cook the fish that we were suppose to catch.

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Plasmid Standard Preparation by Serial Dilution

Following the method of Applied Biosystems we arrived at this computation but still we were not able to get a good standard instead we got a noisy standard. Please help us solved this problem. Please comment here.

How can we improve our standard?

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. 

We encountered problem like this using plasmid standard. Please look at the illustration.

One proposal to solve  problem is the use of MS2 RNA. We still haven't tried it yet.

We did the serial dilution of the samples following the computation by Applied Biosystems. Click the link for computation.

Enzyme Assay for Glutathione Reductase Improved

Glutathione reductase assay GR activity was determined following the procedures of Halliwell and Foyer (1978) with slight modifications, by measuring the decrease in absorbance at 340 nm. The reaction mixture with a total volume of  1 ml contains 100 µl of    0.5 M  potassium phosphate containing 1 mM EDTA (pH 7.8), 810 µl of distilled water, 50 µl of crude extract, 20 µl of 10 mM NADPH in 50 mM potassium phosphate containing 1 mM EDTA (pH 7.8) and 20 µl of 10 mM oxidized glutathione(GSSG) in 10 mM potassium phosphate. The components were added in the 1-mL quartz cuvet   in the same order with GSSG as the last component. Since the enzyme extract was crude, 2 minutes was allotted for the nonspecific oxidation of NADPH before adding the GSSG. The reaction mixture was carefully mixed and read immediately at 340 nm  at an interval time  of 1.5 seconds for a  total time of  300 seconds using the DU 800 Spectrophotometer All measurements were made in triplicates. The activity of the enzyme was calculated using molar absorptivity constant of NADPH (6.2 mM^-1cm^-1). The activity of glutathione reductase was expressed in terms of µmol NADPH oxidized per mg protein per minute.


-Blue Lab