VECTOR NTI

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silver
silver's picture
VECTOR NTI

 I am trying to design primers for soybean rt Pcr and my problem is many cdna sequences are incomplete . to check the primer specificity i need to find the complete sequence of them (the homologs). I heard you can do this on vector NTI. can anyone help me with this.
thankssssssssssssssssssssss....

Ivan Delgado
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Hi Silver,

Hi Silver,

I'm not sure if this suggestion helps, but have you tried BLASTing your partial cDNA sequences to see if you can find larger cDNAs in SoyBase or other soybean DNA databases? 

silver
silver's picture
 Hi Ivan,

 Hi Ivan,
I got these sequences from Glycene max transcriptome atlas. I ll try the one u suggested.
In the meantime, if u know any other SW pls let me know,
cheers,
silver

Ivan Delgado
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Sounds good. I really can't

Sounds good. I really can't think of another resource to find cDNA from soybean, but if you have troubles finding what you need let me know and I'll see if I can be of any additional help.

Cheers

silver
silver's picture
 Hi Ivan,

 Hi Ivan,
I tried the DB u suggested, but I cudnt retrive the sequences, it gave  me the homology anyhw.
This  may be a silly question, but I really dont know the in s and outs of Q PCR.
so I m just wondering, what if the primers we design bind to another gene but this product size is far bigger or (smaller) than the one that we are interested in. What sort of an out put can u expect in the analysis.
If this is ina gel, we can visualize it and identify due to the diff prdct sizes, but can such difference be identified in the q PCR?
Hehe hope this is not THAT silly..

Ivan Delgado
Ivan Delgado's picture
A typical/normal qPCR assay

A typical/normal qPCR assay only amplifies a very short piece of DNA, between 100 and 200 bp. As a result if your primers amplify another amplicon that is a few times bigger, say 1,000 bp, there is a very good chance you would not get any signal in your qPCR assay. 

Of course it all depends on the kind of qPCR conditions you have. If your qPCR has an annealing and extension cycle that is 30 seconds or longer, you may be able to amplify larger fragments. But if you are using a standard aPCR program, with extension and annealing cycle of less than 30 cycles, then fragments that are 1,000 bp or larger will likely not amplify.

Hope this help.

silver
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 Many thanks Ivan..

 Many thanks Ivan..

Biju
Biju's picture
Hi Silver

Hi Silver
One reason for not getting any results in blast could be due to the long length og the cDNA sequence you have used.I suggest you input 150-200 bp sequence around which you want to design the primers.You should also try blastn instead of blast.
Biju