PCR/sequencing

[fizzle123456789]

Hi,

Im a little confused with the results i got maybe someone here could help me on this

basically i did PCR and i got a band on the gel which was approx 225-300bp but when the pcr product it was sanger sequenced i recieved a 401 nucleotide long result which part of the gene for drosha.

sequence adapters maybe?

how could this be, the gel shows a thick band with the middle of the band aprox 262 base pairs.

what could cause this difference in result, it wasnt myself who ran pcr and sequencing but it was i who prepared the mixtures

thank you

[rskr]

Hi,

Im a little confused with the results i got maybe someone here could help me on this

basically i did PCR and i got a band on the gel which was approx 225-300bp but when the pcr product it was sanger sequenced i recieved a 401 nucleotide long result which part of the gene for drosha.

sequence adapters maybe?

how could this be, the gel shows a thick band with the middle of the band aprox 262 base pairs.

what could cause this difference in result, it wasnt myself who ran pcr and sequencing but it was i who prepared the mixtures

thank you

The smear is just that a smear. If you look there should be a distributions of fragment sizes. Could be any number of reasons for this. Differences in electro-chemical potential to just being caught in the some sort of clump.

I think there are usually something like two peaks. One where there was some sort of optimum at the PCR step, and one where the size selection has an optimum. Just my guess...

[fizzle123456789]

I have attached my gel and have also included ladder values.... my pcr product is in lane 2 and control in lane 3 and the ladder in lane 1

and from this PCR product i was handed a 401 long sequence how and why?

thanks