Hello,
It was easy to get the full length of SbBDAH1 (1520 bp) & SbBADH2 (1517 bp) from cDNA using dream taq from thermo scientific, although there were non specific targets and primer dimer, but I could isolate my target genes from the gel. Unexpectedly I couldn't get my target even didn't change any thing of chemicals, kits, primers, cDNA and PCR machine. I had tested cDNA with a reference gene (137 bp) and it works ok but still can't get the full length of the gene.
From the gel pictures it seems that you already got the targeted genes but the quantity is low. you could do another round of PCR from the previous PCR sample to further increase the targeted genes,
you could also try to use gene specific primer during the reverse transcriptase to amplify the number of targeted cdna to increase the input.
if this what you ask.
- Your amplifications seem inefficient and non specific
- You might to check your PCR conditions
- In addition, you seem to have what might be primer dimers
- That being so, this would greatly reduce the efficiency of your PCR reactions and impact particularly on formation of your larger product
- You might want to redesign primers paying particular regard to avoidance of hair pin loops, dimer formation etc
- To that end I have provided an SOP I sometimes give to students explaining the principles and practice of good primer design
- You should also make sure that your target is not top heavy in A/T residues and G/C: The former can reduce the replication efficiency by causing your polymerase to drop off during replication; the latter can block replication due to secondary structure
- Regardless, for targets over 1kb, apart from making good primers I would:
Wahyu Handoko Thanks for your answer and I have also 2 question depending on your comment:
1. How many microliters shold I take in the re-PCR reaction?
2. Do you mean specific gene primer instead of Oligo in cDNA synthesis?
Laurence Stuart Dawkins-Hall Thank you so much for your answer and the attached file it really helps, I would to ask you about annealing time, I used to made it 45 sec, is that Ok?
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