Hi!
Are these bands primer dimers or not?
lanes 1 -> 7: samples
lane 8: negative control
lane 9: generuler 100bp plus (lowest band of ladder = 100bp)
lane 10-> 16: sample = 1 µl from PCR products in lanes 1-> 7 with nested primers
lane 17: negative control (sample = 1 µl from lane 8)
Can anyone help me with the interpretation of this gel?
thank you!
Britt
It is a 5' RACE PCR, so the fragment should be as big as possible, depending on the length of the generated cDNA fragments.
I know that these bands are not the bands I need, but for troubleshooting purposes I want to know if there is amplification of very short fragments, or no amplification at all. In the first case it's the cDNA that is the problem, in the second case it's the PCR conditions or the primers.
I am not familiar with RACE PCR, but I think that only lane 8 shows primer dimers. These are most often smaller than 100bp. The others show product above 100bp.
Also, there is a smear visible what might indicate fragmented cDNA...
You should check the product size. If it corresponds, than no problem. Otherwise, your protocol might not be optimized.
@Sabine
I ran my cDNA samples on a 1.5% agarose gel and the result is a smear (see attachment). Do you think this is why I have no or very short 5'RACE amplified fragments?
the cDNA is made using superscript III reverse transcriptase and a gene specific primer
5' RACE PCR frequently causes a smear; in my opinion there is amplification of very short fragments. As an option, I would use less input in the first PCR, sometimes this helps; you might make a dilutions serious; and as you said I would also try other primer sets; in my opinion your cDNA samples looks ok and highly concentrated, therefore, my suggestion to use less input.
Hi Britt,
I am more less familiar with RACE PCR and my opinion is that your PCR conditions has to be optimized or even you have to chose new primers. If you need more info you can write me; Good luck!
Hi Britt,
Sometimes, we solved our resolution problems with acrilamide gels and silver staining. Agarose gels are pooly resolutive, so you need a more precise idea of the product size. Proper controls are also required to detect specific amplification, i.e. minus RT, to determine the band specificity or TAP treated samples to discriminate between processed and primary transcripts.
I followed different strategies for 5' RACE and most of the times you do not observe a clear band. Usually, we also clone and sequence those bands we think corresponds to the expected products. Anyway, I should recomend you to adjust the nested PCR conditions.
Good luck!
If it is 5' RACE you would have taken gene specific anchored Primers sp1, sp2 & sp3 . just try to check them if they are giving bands in their product size difference than you have to optimize this reactions because here you are getting smearing. and you could also use the 50 bp ladder in 2 % agarose . All the best
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