Currently conducting cDNA amplification during a Tag-Seq (RNA-Seq) protocol. In the gel attached, all wells with 'D' should be cDNA with smears, all others should not because they are controls missing certain oligos during this test PCR that was run. The picture was edited and slight smears faintly appear. These should be amplified cDNA but look like junk because they appear in every well and should not.
We have already troubleshooted with template concentrations, reagent quality and batch, used 2 thermocyclers, different sets of polymerases, tested cDNA already synthesized by various library preps and results have appeared similar in each situation. Any suggestions would be greatly appreciated!
Hi De,
You need to provide more information to get a good answer. Why do you expect a smear from your PCR? A 'standard' cDNA amplification, targeting one transcript, should normally generate a single PCR band. What is the expected size (range) of products? What are the sizes of your DNA markers?
My impression from your gel is that in none of your reactions have any visible PCR product, with the possible exception of the lane with number 20 for the D-group with 19 cycles (bottom right; following your logic for loading, I assume this is group D, even though it's marked C).
Unless you expect your gene to be extremely highly expressed, 19 PCR cycles (or even less) is not very much. You might want to extend your program with 5-10 cycles.
Moreover, you seem to have loaded high molecular weight DNA ladders. If you expect multi-kb products, you will need (1) to use a longer extension-time for your PCR and (2) possibily the use of specialized Taq-polymerases for long products.
I hope this helps you on your way,
Daan
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