The expect PCR products are the bottom one and the middle one (124 bp difference in size due to additional exon).
Now we don't understand what is the top one (around 850 bp) that is so strong?
I purified the top one from gel (sample1 and 4 from first PCR). GelPurification is the image of gel I used for gel purification and use purified product as template run a secondary PCR. SecondaryPCR shows the gel image of second PCR results and although I only purified top band, I still got lower 2 bands using same primer (F173+R819).
My thought is that the relative large number of top band somehow prevents very few of lower 2 fragments from migrated to appropriate place and may likely trapped with top bands. That is why I still got traces of 2 lower bands at secondary PCR because they also got purified with top band.
I am very sure I DID NOT touch any of lower bands because I run another longer gel for gel purification and the distance between top and middle band is at least 3mm I donít think anyone can miss that (as shown in GelPurification).
My boss did not buy my idea.
Is my idea wrong? If I am wrong, and I think I did not cut of any lower bands, what produce those lower 2 bands during second PCR?
During second PCR, I also try a difference primer pair (F137+R573) that the reverse primer move about 100 bp (near 250 bp of include 124 bp insert) upstream which will not detect additional 124 bp fragment. The result shows the bottom band shift downward about 150 bp and no middle band identified; however, the top band remains at same position even use gel purified product (F137+R819 PCR product).
I also send purified PCR product from first PCR for direct sequencing and result indicates this purified product contain 2 equally strong templates (as shown in Sequencing). The sequence alignment and fragment search suggest that this band probably has nothing to do with our target gene (2 lower bands).
So what is this upper band? And how is that get there?
My boss simply thinks I screw up during gel purification; and the evidence are I got lower bands during secondary PCR and multi template of sequencing.
I try to explain myself (stupid thing to do to a big professor), and my boss end our argument by giving me 2 weeks notice. I am going to leave anyway, I actually glad it end sooner.
Did I screw up during gel purification as my boss said? If I did, can anyone tell me what went wrong and how can I improve it?
Edited by wuxx0153, 10 May 2009 - 10:48 AM.