cloning from cDNA but got a much shorter PCR product (View forum version)



kblee

Posted 25 September 2012 - 09:17 AM

Hi everyone,

Recently I need to clone a gene (around 1000bp) from cDNA (commercial normal human gastro),
the gene is around 1000bp but I only have two different bands size 400bp,600bp respectively.

The gene in the cDNA panel is not abundant, likes GAPDH in RTPCR is Ct20 but my target gene reach Ct30.
Any idea what's happening of my cloning and any suggestion for me?

Thanks in advance Posted Image

bob1

Posted 25 September 2012 - 12:48 PM

I take it that you are cloning from PCR - how are you doing this - linker ligation? TA cloning? tagged primers?

Have you optimised the PCR for a 1000 bp band before trying the cloning?

kblee

Posted 26 September 2012 - 12:19 AM

I take it that you are cloning from PCR - how are you doing this - linker ligation? TA cloning? tagged primers?

Have you optimised the PCR for a 1000 bp band before trying the cloning?


Yes I am cloning from PCR but my PCR product size doesn't match the size which search from UCSC.
The translated region of this gene is around 1000bp, but using the primer which carry restriction enzyme site to amplify cDNA, only 600bp and 400bp band size.
How can I amplify the gene from cDNA?

Thanks!

bob1

Posted 26 September 2012 - 01:20 AM

Have you tried changing the annealing temperature, Mg concentration, template concentration, extension times,..?

PlantS_Student

Posted 26 September 2012 - 10:08 AM

Assuming the RNA quality was good and you are using a good enzyme, have you tested the primer pair on genomic DNA to check that the primer pair works at the Ta you are using? I just amplified a 2 KB gene from cDNA recently (not directly RT PCR product) using the Superscript III and oligo dT primer and it went quite smoothly.