4 replies to this topic

[niveda]

Hi all,
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
niveda

Attached Thumbnails

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[Rsm]

To me it looks like you have at least two different sequences overlapping. You do a PCR and then sequence straight from your genomic DNA? So if you start with heterogenous DNA, you'll end up with two signals... Maybe you can do an intermediate cloning step? Like clone your PCR product into TA vector, put into bacteria and sequence the insert. That way you'll have only one DNA sequence and one peak.

Cheers,

Minna

[Maddie]

Can you give us the info? (per base)
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.

[mdfenko]

you should clean the pcr product before sequencing. carried over components (especially primers and dntps) will interfere with sequencing.

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.

[ElHo]

did you check your pcr products by gel electrophoresis? Maybe you get some byproducts.