Dear All,
I am now doing a research about ABC transporter protein in oil palm. By taking AtABCG40 gene from Arabidopsis as my reference gene, I've blast it with oil palm database. I need to get the full sequence of the gene for the functional analysis later. I tried run PCR by designing the primer in normal way but failed to get any band. Can anyone suggest me what should i do? Some suggest me to do contig with my my sequence but i don't have any idea about that. Hope you guys can help me out. Thank you.
I agree completely with Adam, plus if your gene of interest is too big and you're unable to get a single PCR product, then you can consider to amplify overlapping fragments and fuse them together through overlapping extension PCR or even Gibson Assembly cloning.
Hi Farah,
The PCR not working could be down to lots of reasons. Things you should consider are the size of the region you are trying to amplify which influences your enzyme choice, extension time and temperature (longer regions might require specialised long range enzymes), GC content, region length and enzyme choice can influence your denaturation step (temperature, time and whether you might want to consider using a co-solvent). The sequence variability of your primer regions can obviously play a big part. All of these (an other things) can stop you getting any product at all. Check you are using the optimal conditions for the reaction.
I might be wrong, but I don't think a contig would be helpful (that's just joining sequences together). What they may mean, and what would be helpful, is finding as many deposited sequences (from that oil palm gene), performing and alignment and making sure your primers are not sat over any polymorphisms which could stop the PCR working.
hope that helps!
Hi Farah,
Check the size of your gene first. There could be many reasons for the failed result. Design your primers properly avoiding the self complementary or primer dimer situations.
I agree completely with Adam, plus if your gene of interest is too big and you're unable to get a single PCR product, then you can consider to amplify overlapping fragments and fuse them together through overlapping extension PCR or even Gibson Assembly cloning.
First find out the conserved region and design the primer having at least 23-25 nucleotide that will increase the specificity and if you want to synthesize the complete cDNA sequence. First select the conserved region and based on that region design primers for RACE (rapid amplification of cDNA) for which kit along with protocol is available. Designing of primer is critical, so be careful and do 5' and 3' race, then you will get your whole cDNA sequence. After getting 5' and 3' sequence, remove the overlapping part and that will result in complete CDS of your sequence.
Thank you guys for the suggestions. Mr. Paolo, about the overlapping fragment, can you suggest me any software can be used to generate those fragments? i've found a website http://pcrsuite.cse.ucsc.edu/Overlapping_Primers.html that can generate the overlapping primers. is that overlapping fragment is similar as overlapping primer? thanks in advance
I loaded a simple and self explaining image about what I mean when I say "overlapping PCR fragments".
You can design your own primer couples in the way that you could have 40-100 overlapping bases between your fragment's sequences. This is the correct range of overlap for Gibson Assembly and also a good range for overlap extension PCR.
About primers-design tools you can use primer3 or PrimerBLAST as well. There are other tool but I usually design my own primers by eye-screening the target sequence and test them via PrimerBLAST + IDT OligoAnalyzer.
you i think you must blast your gene of interest in NCBI blast , here is the complete sequence of your gene you may find it. and then blast NCBI primers and select the most relevant 100% efficiency, E value
Hi Farah, I believe there are a number of primer design websites where you can get your primer characteristics properly done. Try primer3plus. However as others have suggested you need to be sure your PCR conditions are well set. atimes this requires optimising by running a gradient PCR or simply trying out different annealing temperatures. I wish you the best.
Most eukaryotic genomic DNA gene coding sequences have introns, in that case you need the full-length cDNA not genomic DNA to clone the gene. Check the database if there are introns in your gene coding region or there is any cDNA clone. If there is no cDNA clone, your gene coding sequence might a predicted version which is not guaranteed 100% right. The quick way to get the gene is by gene synthesis if there is no cDNA clone existed.
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