Help Help, sequencing not working
Posted 30 July 2010 - 01:40 AM
I have been doing this seq standardization from march, was not getting anything , lately i have changed my clean up protocol- suddenly it started coming , but 12/17 samples are giving a noise signal , please help, any small suggestion will do ,
More-info: i am working on genomic sequences, searching for new SNPs and mutations, my genes are GC rich,
i do a pcr in 25ul, take 1ul of this and and add 10picomoles of proimer ,1ul ABI mix, 2ul of buffer and 5ul of water.
clean up is 10ul water+2ul 125mM EDTA+2ulSodium acetate+50ul chilled ethanol, 15 mins in ice, 15min spin, 250ul 70% ethanol wash, air drying.(SEQ file attached)
Hope you will help
Eagerly waiting, please
Posted 30 July 2010 - 01:47 AM
Posted 30 July 2010 - 09:15 AM
I mean what is the intensity of these bands?
If you use sequencher, you have a button called "get info".I
I ask because your data could be due to too much or not enough sequencing product.
Edited by Maddie, 30 July 2010 - 01:13 PM.
Posted 30 July 2010 - 12:32 PM
if you don't then you have to dilute at least 10x but have >1fmol/ul product after dilution.
Edited by mdfenko, 30 July 2010 - 12:37 PM.
genius does what it must
i do what i get paid to do
Posted 03 August 2010 - 01:04 AM