PCR amplification problem

[bam pandit]

thats nice to hear priyanka... looks like you've monitored all the factors correctly... and to trace the bad quality in agarose and ethanol is really an acievement, as in, these factors may never strike at all when the results are not showing up... great going... all the best... will let you know the progress on my problem as and when it happens

[Priyanka]

Yeah I am relly sorry to bug you again I am attaching gel pic along.. pls tell me whcih band is what..... There are way to many bands to comprehend....The first one`s DNA which ones 28,16s etc... please tell me your interpretation

[bam pandit]

hey sorry for the late reply priyanka. i saw the picture you have attached. it appears to a be a not so good RNA preparation. the first band after the genomic DNA band is the 28S band, though its very light (should be thrice as bright as the band below it)... the band below it would be the 18S and the bands further down the line will comprise of the other small RNAs (that you come across in plants....say from chloroplasts)

Also i would like to ask whether you had mixed the EtBr in the gel or you had stained it later... because if you had mixed EtBr in the gel then it appears that you didn't mix it well.. ... take care about that the next time. plus in the gel you should take more care when pulling out the comb.

[Priyanka]

i had mixed etbr in the gel itself....(how could you undertsand that it was not mixed properly...this would improove my comprehension...)..and why do u think that 28s band is not as bright as it should be...(any suggestions for that one...)....i knew 28s and 18 s bands but i was hoping that you could tell me what those multiple bands are of .... thanx for the help buddy..what happened to your gene.. any luck..
Cheers

[Muthukumaravel.S]

I am getting smearing of bands. my product is 293 kb.

[bam pandit]

hmmm... are you sure about the template'scomdition, because 300 bp isn't a very large fragment to amplify, if you are using a cDNA, it shouldn't be very old, preferrably freshly made.
you should chk the annealing temperature, and whether the primers have similar melting temperatures. Mg ion conc is also important, some PCR buffers need it to be added to them, chk wih that too.
let me know the results when you repeat the expt with modifications

[halramin]

The RNA preparation is badly degraded from the gel pic you attached. I would redo it. But for a 3.5 Kb fragment, I think the error rate for Taq is 65%. I use Phusion High-fedility polymerase. In an HF buffer it's error rate is only about 1.5% for a 3.5 Kb fragment.