Marjan, there are several tools (BioEdit, Dnasis Max) which help you to find regions with high homology. However, you also can do this via visual screening through the sequence alignment. I assume you know which region you want to amplify. In this sequence region you look for well conserved region.
If you design the primers, you should follow some basic rules:
1.) The length of a primer sequence should be around 20 bp
2.) The GC content should be around 50-55%
3.) The primer should end on the 3' end either with a G or a C
4.) The forward and reverse primer shouldn't be identical (very unlikely)
5.) The melting temperature of both primers should be roughly the same and should be around 50-60 °C
Good luck
Ijad
Marjan, there are several tools (BioEdit, Dnasis Max) which help you to find regions with high homology. However, you also can do this via visual screening through the sequence alignment. I assume you know which region you want to amplify. In this sequence region you look for well conserved region.
If you design the primers, you should follow some basic rules:
1.) The length of a primer sequence should be around 20 bp
2.) The GC content should be around 50-55%
3.) The primer should end on the 3' end either with a G or a C
4.) The forward and reverse primer shouldn't be identical (very unlikely)
5.) The melting temperature of both primers should be roughly the same and should be around 50-60 °C
Good luck
Ijad
You can take the region with maximum homology, and design primers from the sequence.
Marjan, there are several tools (BioEdit, Dnasis Max) which help you to find regions with high homology. However, you also can do this via visual screening through the sequence alignment. I assume you know which region you want to amplify. In this sequence region you look for well conserved region.
If you design the primers, you should follow some basic rules:
1.) The length of a primer sequence should be around 20 bp
2.) The GC content should be around 50-55%
3.) The primer should end on the 3' end either with a G or a C
4.) The forward and reverse primer shouldn't be identical (very unlikely)
5.) The melting temperature of both primers should be roughly the same and should be around 50-60 °C
Good luck
Ijad
Thanks I know the basic knowledge for design primer but I do not know how take maximum homology.I read the help manuale of software . What sould we do with gap part! what is penalty or penalise in gap open, gap extension, gap distance !
Is there any soft ware to delet gaps and combine and detrmine the best homology when we comparing several sequences.
Marjan,
if you do an alignment, sometimes gaps has to be introduced to make the alignment work. Gaps are the most expensive operations. This is was is meant with gap penalty.
-Ijad
If we have a primer of complete sequence of gene of interest but the primer is so long for examle(6000 -7000) lenghth and we need shorter primer.What should we do for getting shorter primer?
In addition, if we have some partial primer, that ones can help us to get complete CDs?
What is your suggestion?
What sould we do with gaps after alignment?
sorry if my questions are elementary.
How manually editing multiple alignment? What factors are important for editing?
Hi.. manual alignment is an important part of Multiple seq Alignment. First do the alignment by using some efficient softwares. then watch the alignment carefully. Use recently developed softwares for alignment. MAFFT and MUSCLE are more efficient than the clustalw. MAFFT and MUSCLE are available online. Save the output in .txt format. Then you can use that text file for manual alignment. In manual alignment try to realign the unaligned regions. Remove Insertions, deletions and most unaligned regions. U can do manual alignment in MEGA 5 or some other alignment softwares.
Best wishes
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