RNA实验

The pSico and pSicoR Design and Cloning

2024-11-06 RNA实验加入收藏

pSico Oligo DesignClick on the Mac OSX program pSicoOligomaker 1.5 to select and

pSico Oligo Design

Click on the Mac OSX program pSicoOligomaker 1.5 to ｓｅｌｅｃｔ and design oligos for pSico and plentilox3.7.

To operate it, simply paste your sequence in the "sequence" window, give it a name (optional), ｓｅｌｅｃｔ a cutoff value (from -2 to 9) and click "search". The program will list in the right window all the potential 19-mers with a score equal or higher than the cutoff. The score is given based on a set of criteria published in Nature Biotech. by Reynolds et al. in 2004. According to the authors, a 19-mer with a value higher than 6 has approximately 90% chance of achieving silencing of the target mRNA.

You can ｓｅｌｅｃｔ all targets and paste them in Word or Excel to store the result. In addition, if you type or paste a 19-mer in the field "target" and click on "make oligos" the program returns the sequence of the forward and reverse oligos (both in the 5' to 3' orientation) that have to be ordered to clone in pSico. The button "order" directs you to the web page we use to order oligos. The button "settings" lets you change the criteria to design the oligos (loop sequence and length, overhangs, etc) so that it can be used to design oligos for other vectors.

"Clear sequence" tells the program to remove all illicit characters, including spaces and "end of line", from the sequence. In addition, it converts the sequence to all caps.

In case you don't have a Mac, or you prefer to design your oligos manually, here is an example:

19mer: GTAGCTTAGCGTCGGAGCT

sense oligo:

5'-TGTAGCTTAGCGTCGGAGCT-TTCAAGAGA-AGCTCCGACGCTAAGCTACTTTTTTC

antisense oligo:

5'-TCGAGAAAAAAGTAGCTTAGCGTCGGAGCT-TCTCTTGAA-AGCTCCGACGCTAAGCTACA

How to clone into pSico and pSicoR:

We order 5’ phosphorylated, PAGE purified oligos designed using the program PSICOLIGOMAKER 1.5.

Oligos are resuspended in distilled water to a final concentration of 100µM.

Oligos annealing:

Mix:

-- ddH2O 23µl